Label-free high-throughput optical technique for detecting biomolecular interactions

ABSTRACT

Methods and compositions are provided for detecting biomolecular interactions. The use of labels is not required and the methods can be performed in a high-throughput manner. The invention also provides optical devices useful as narrow band filters.

PRIORITY

This is a divisional Ser. No. 09/930,352 filed Aug. 15, 2001 now U.S.Pat. No. 7,094,595, which claims the benefit of application Ser. No.60/244,312, filed Oct. 30, 2000; U.S. Provisional application Ser. No.60/283,314, filed Apr. 12, 2001; and U.S. Provisional application Ser.No. 60/303,028, filed Jul. 3, 2001.

TECHNICAL AREA OF THE INVENTION

The invention relates to compositions and methods for detectingbiomolecular interactions. The detection can occur without the use oflabels and can be done in a high-throughput manner. The invention alsorelates to optical devices.

BACKGROUND OF THE INVENTION

With the completion of the sequencing of the human genome, one of thenext grand challenges of molecular biology will be to understand how themany protein targets encoded by DNA interact with other proteins, smallmolecule pharmaceutical candidates, and a large host of enzymes andinhibitors. See e.g., Pandey & Mann, “Proteomics to study genes andgenomes,” Nature, 405, p. 837-846, 2000; Leigh Anderson et al.,“Proteomics: applications in basic and applied biology,” Current Opinionin Biotechnology, 11, p. 408-412, 2000; Patterson, “Proteomics: theindustrialization of protein chemistry,” Current Opinion inBiotechnology, 11, p. 413-418, 2000; MacBeath & Schreiber, “PrintingProteins as Microarrays for High-Throughput Function Determination,”Science, 289, p. 1760-1763, 2000; De Wildt et al., “Antibody arrays forhigh-throughput screening of antibody-antigen interactions,” NatureBiotechnology, 18, p. 989-994, 2000. To this end, tools that have theability to simultaneously quantify many different biomolecularinteractions with high sensitivity will find application inpharmaceutical discovery, proteomics, and diagnostics. Further, forthese tools to find widespread use, they must be simple to use,inexpensive to own and operate, and applicable to a wide range ofanalytes that can include, for example, polynucleotides, peptides, smallproteins, antibodies, and even entire cells.

Biosensors have been developed to detect a variety of biomolecularcomplexes including oligonucleotides, antibody-antigen interactions,hormone-receptor interactions, and enzyme-substrate interactions. Ingeneral, biosensors consist of two components: a highly specificrecognition element and a transducer that converts the molecularrecognition event into a quantifiable signal. Signal transduction hasbeen accomplished by many methods, including fluorescence,interferometry (Jenison et al., “Interference-based detection of nucleicacid targets on optically coated silicon,” Nature Biotechnology, 19, p.62-65; Lin et al., “A porous silicon-based optical interferometricbiosensor,” Science, 278, p. 840-843, 1997), and gravimetry (A.Cunningham, Bioanalytical Sensors, John Wiley & Sons (1998)).

Of the optically-based transduction methods, direct methods that do notrequire labeling of analytes with fluorescent compounds are of interestdue to the relative assay simplicity and ability to study theinteraction of small molecules and proteins that are not readilylabeled. Direct optical methods include surface plasmon resonance (SPR)(Jordan & Corn, “Surface Plasmon Resonance Imaging Measurements ofElectrostatic Biopolymer Adsorption onto Chemically Modified GoldSurfaces,” Anal. Chem., 69:1449-1456 (1997), (grating couplers (Morhardet al., “Immobilization of antibodies in micropatterns for celldetection by optical diffraction,” Sensors and Actuators B, 70, p.232-242, 2000), ellipsometry (Jin et al., “A biosensor concept based onimaging ellipsometry for visualization of biomolecular interactions,”Analytical Biochemistry, 232, p. 69-72, 1995), evanascent wave devices(Huber et al., “Direct optical immunosensing (sensitivity andselectivity),” Sensors and Actuators B, 6, p. 122-126, 1992), andreflectometry (Brecht & Gauglitz, “Optical probes and transducers,”Biosensors and Bioelectronics, 10, p. 923-936, 1995). Theoreticallypredicted detection limits of these detection methods have beendetermined and experimentally confirmed to be feasible down todiagnostically relevant concentration ranges. However, to date, thesemethods have yet to yield commercially available high-throughputinstruments that can perform high sensitivity assays without any type oflabel in a format that is readily compatible with the microtiterplate-based or microarray-based infrastructure that is most often usedfor high-throughput biomolecular interaction analysis. Therefore, thereis a need in the art for compositions and methods that can achieve thesegoals.

SUMMARY OF THE INVENTION

It is an object of the invention to provide compositions and methods fordetecting binding of one or more specific binding substances to theirrespective binding partners. This and other objects of the invention areprovided by one or more of the embodiments described below.

One embodiment of the invention provides a biosensor comprising: atwo-dimensional grating comprised of a material having a high refractiveindex, a substrate layer that supports the two-dimensional grating, andone or more specific binding substances immobilized on the surface ofthe two-dimensional grating opposite of the substrate layer. When thebiosensor is illuminated a resonant grating effect is produced on thereflected radiation spectrum. The depth and period of thetwo-dimensional grating are less than the wavelength of the resonantgrating effect.

Another embodiment of the invention provides an optical devicecomprising a two-dimensional grating comprised of a material having ahigh refractive index and a substrate layer that supports thetwo-dimensional grating. When the optical device is illuminated aresonant grating effect is produced on the reflected radiation spectrum.The depth and period of the two-dimensional grating are less than thewavelength of the resonant grating effect.

A narrow band of optical wavelengths can be reflected from the biosensoror optical device when the biosensor is illuminated with a broad band ofoptical wavelengths. The substrate can comprise glass, plastic or epoxy.The two-dimensional grating can comprise a material selected from thegroup consisting of zinc sulfide, titanium dioxide, tantalum oxide, andsilicon nitride.

The substrate and two-dimensional grating can optionally comprise asingle unit. The surface of the single unit comprising thetwo-dimensional grating is coated with a material having a highrefractive index, and the one or more specific binding substances areimmobilized on the surface of the material having a high refractiveindex opposite of the single unit. The single unit can be comprised of amaterial selected from the group consisting of glass, plastic, andepoxy.

The biosensor or optical device can optionally comprise a cover layer onthe surface of the two-dimensional grating opposite of the substratelayer. The one or more specific binding substances are immobilized onthe surface of the cover layer opposite of the two-dimensional grating.The cover layer can comprise a material that has a lower refractiveindex than the high refractive index material of the two-dimensionalgrating. For example, a cover layer can comprise glass, epoxy, andplastic.

A two-dimensional grating can be comprised of a repeating pattern ofshapes selected from the group consisting of lines, squares, circles,ellipses, triangles, trapezoids, sinusoidal waves, ovals, rectangles,and hexagons. The repeating pattern of shapes can be arranged in alinear grid, i.e., a grid of parallel lines, a rectangular grid, or ahexagonal grid. The two-dimensional grating can have a period of about0.01 microns to about 1 micron and a depth of about 0.01 microns toabout 1 micron.

The one or more specific binding substances can be arranged in an arrayof distinct locations and can be immobilized on the two-dimensionalgrating by physical adsorption or by chemical binding. The distinctlocations can define a microarray spot of about 50-500 or 150-200microns in diameter. The one or more specific binding substances can bebound to their binding partners. The one or more specific bindingsubstances can be selected from the group consisting of nucleic acids,polypeptides, antigens, polyclonal antibodies, monoclonal antibodies,single chain antibodies (scFv), F(ab) fragments, F(ab′)₂ fragments, Fvfragments, small organic molecules, cells, viruses, bacteria, andbiological samples. The biological sample can be selected from the groupconsisting of blood, plasma, serum, gastrointestinal secretions,homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum,cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lunglavage fluid, semen, lymphatic fluid, tears, and prostatitc fluid. Thebinding partners can be selected from the group consisting of nucleicacids, polypeptides, antigens, polyclonal antibodies, monoclonalantibodies, single chain antibodies (scFv), F(ab) fragments, F(ab′)₂fragments, Fv fragments, small organic molecules, cells, viruses,bacteria, and biological samples. The biosensor can further comprise anantireflective dielectric coating on a surface of the substrate oppositeof the two-dimensional grating. The biosensor can comprise anantireflective physical structure that is embossed into a surface of thesubstrate opposite of the two-dimensional grating, such as a motheyestructure. The biosensor can comprise an internal surface of aliquid-containing vessel. The vessel is selected from the groupconsisting of a microtiter plate, a test tube, a petri dish and amicrofluidic channel. The biosensor can be attached to a bottomlessmicrotiter plate by a method selected from the group consisting ofadhesive attachment, ultrasonic welding and laser welding.

Another embodiment of the invention provides a detection systemcomprising a biosensor or optical device of the invention, a lightsource that directs light to the biosensor or optical device, and adetector that detects light reflected from the biosensor. The detectionsystem can comprise a fiber probe comprising one or more illuminatingoptical fibers that are connected at a first end to the light source,and one or more collecting optical fibers connected at a first end tothe detector, wherein the second ends of the illuminating and collectingfibers are arranged in line with a collimating lens that focuses lightonto the biosensor or optical device. The illuminating fiber and thecollecting fiber can be the same fiber. The light source can illuminatethe biosensor from its top surface or from its bottom surface.

Even another embodiment of the invention provides a method of detectingthe binding of one or more specific binding substances to theirrespective binding partners. The method comprises applying one or morebinding partners to a biosensor of the invention, illuminating thebiosensor with light, and detecting a maxima in reflected wavelength, ora minima in transmitted wavelength of light from the biosensor. Whereone or more specific binding substances have bound to their respectivebinding partners, the reflected wavelength of light is shifted.

Still another embodiment of the invention provides a method of detectingthe binding of one or more specific binding substances to theirrespective binding partners. The method comprises applying one or morebinding partners to a biosensor of the invention, wherein the biosensorcomprises a two-dimensional grating that is coated with an array ofdistinct locations containing the one or more specific bindingsubstances. Each distinct location of the biosensor is illuminated withlight, and maximum reflected wavelength or minimum transmittedwavelength of light is detected from each distinct location of thebiosensor. Where the one or more specific binding substances have boundto their respective binding partners at a distinct location, thereflected wavelength of light is shifted.

Yet another embodiment of the invention provides a method of detectingactivity of an enzyme. The method comprises applying one or more enzymesto a biosensor of the invention, washing the biosensor, illuminating thebiosensor with light, and detecting reflected wavelength of light fromthe biosensor. Where the one or more enzymes have altered the one ormore specific binding substances of the biosensor by enzymatic activity,the reflected wavelength of light is shifted.

Another embodiment of the invention provides a biosensor comprising asheet material having a first and second surface, wherein the firstsurface defines relief volume diffraction structures, a reflectivematerial coated onto the first surface of the sheet material, and one ormore specific binding substances immobilized on the reflective material.Still another embodiment of the invention provides an optical devicecomprising a sheet material having a first and second surface, whereinthe first surface defines relief volume diffraction structures, and areflective material coated onto the first surface of the sheet material.The biosensor or optical device reflects light predominantly at a firstsingle optical wavelength when illuminated with a broad band of opticalwavelengths. The biosensor reflects light at a second single opticalwavelength when the one or more specific binding substances areimmobilized on the reflective surface. The reflection at the first andsecond optical wavelengths results from optical interference. Thebiosensor can reflect light at a third single optical wavelength whenthe one or more specific binding substances are bound to theirrespective binding partners. The reflection at the third opticalwavelength results from optical interference. The depth and period ofthe relief volume diffraction structures can be less than the resonancewavelength of the light reflected from the biosensor. The depth of therelief volume diffraction structures can be about 0.01 microns to about1 micron. The period of the relief volume diffraction structures can beabout 0.01 microns to about 1 micron. The relief volume diffractionstructures can be about 0.5 microns to about 5 microns in diameter.

Even another embodiment of the invention provides a biosensor comprisinga two-dimensional grating having a first and a second surface comprisedof an optically transparent material that conducts electricity. Thefirst surface of the grating is coated with an electrical insulator, andthe second surface of the grating is deposited on a substrate. When thebiosensor is illuminated, a resonant grating effect is produced on thereflected radiation spectrum. The depth and the period of the gratingare less than the wavelength of the resonant grating effect. Two or moreseparate grating regions can be present on the same substrate. Anelectrically conducting trace to each separate grating region of thesubstrate can be present. The conducting trace can be connected to avoltage source. One or more specific binding substances can be bound toeach separate grating region of the substrate.

Yet another embodiment of the invention provides a method of measuringthe amount of binding partners in a test sample. One or more bindingpartners are immobilized to the biosensor described above. An electricalcharge is applied to the electrically conducting traces. The biosensoris illuminated with light and the reflected wavelength of light isdetected from the biosensor. Where the one or more specific bindingsubstances have bound to their respective binding partners, thereflected wavelength of light is shifted. A reversed electrical chargecan be applied to the electrically conducting traces before illuminatingthe biosensor with light.

Still another embodiment of the invention provides a method of detectingthe binding of one or more specific binding substances to theirrespective binding partners. The method comprises illuminating abiosensor of the invention with light, detecting reflected wavelength oflight from the biosensor, applying a test sample comprising one or morebinding partners to the biosensor, illuminating the biosensor withlight, and detecting reflected wavelength of light from the biosensor.The difference in wavelength of light is a measurement of the amount ofone or more binding partners in the test sample.

Another embodiment of the invention provides a detection systemcomprising a biosensor of the invention, a light source that directslight at the biosensor, and a detector that detects light reflected fromthe biosensor. A first illuminating fiber probe having two ends isconnected at its first end to the detector. A second collection fiberprobe having two ends is connected at its first end to the light source.The first and second fiber probes are connected at their second ends toa third fiber probe, which acts as an illumination and collection fiberprobe. The third fiber probe is oriented at a normal angle of incidenceto the biosensor and supports counter-propagating illuminating andreflecting optical signals.

Even another embodiment of the invention provides a detection systemcomprising a biosensor of the invention, a light source that directslight at the biosensor, and a detector that detects light reflected fromthe biosensor. An illuminating fiber probe is connected to the lightsource and is oriented at a 90 degree angle to a collecting fiber probe.The collecting fiber probe is connected to the detector, wherein lightis directed through the illuminating fiber probe into a beam splitterthat directs the light to the biosensor. Reflected light is directedinto the beam splitter that directs the light into the collecting fiber.

Still another embodiment of the invention comprises a method ofimmobilizing one or more specific binding substances onto a biosensor ofthe invention. The method comprises activating the biosensor with amine,attaching linker groups to the amine-activated biosensor, and attachingone or more specific binding substances to the linker groups. Thebiosensor can be activated with amine by a method comprising immersingthe biosensor into a piranha solution, washing the biosensor, immersingthe biosensor in 3% 3-aminopropyltriethoxysilane solution in dryacetone, washing the biosensor in dry acetone, and washing the biosensorwith water. A linker can be selected from the group consisting of amine,aldehyde, N,N′-disuccinimidyl carbonate, and nickel.

Yet another embodiment of the invention provides a method of detectingthe binding of one or more specific binding substances to theirrespective binding partners. The method comprises applying one or morebinding partners comprising one or more tags to a biosensor of theinvention, illuminating the biosensor with light, and detectingreflected wavelength of light from the biosensor. Where the one or morespecific binding substances have bound to their respective bindingpartners, the reflected wavelength of light is shifted. The one or moretags can be selected from the group consisting of biotin, SMPT, DMP,NNDC, and histidine. The one or more tags can be reacted with acomposition selected from the group consisting of streptavidin,horseradish peroxidase, and streptavidin coated nanoparticles, beforethe step of illuminating the biosensor with light.

Another embodiment of the invention provides a biosensor compositioncomprising two or more biosensors of the invention, where the biosensorsare associated with a holding fixture. The biosensor composition cancomprise about 96, about 384, or about 50 to about 1,000 individualbiosensors. Each of the two or more biosensors can comprise about 25 toabout 1,000 distinct locations. Each biosensor can be about 1 mm² toabout 5 mm², or about 3 mm². The holding fixture can hold each biosensorsuch that each biosensor can be placed into a separate well of amicrotiter plate.

Even another embodiment of the invention provides a biosensorcomposition comprising one or more biosensors of the invention on a tipof a multi-fiber optic probe. The one or more biosensors can befabricated into the tip of the probe or can be attached onto the tip ofthe probe.

Still another embodiment of the invention provides a method of detectingbinding of one or more specific binding substances to their respectivebinding partners in vivo. The method comprises inserting the tip of thefiber optic probe described above into the body of a human or animal,illuminating the biosensor with light, and detecting reflectedwavelength of light from the biosensor. If the one or more specificbinding substances have bound to their respective binding partners, thenthe reflected wavelength of light is shifted.

Yet another embodiment of the invention provides a detection systemcomprising a biosensor of the invention, a laser source that directs alaser beam to a scanning mirror device, wherein the scanning mirrordevice is used to vary the laser beam's incident angle, an opticalsystem for maintaining columination of the incident laser beam, and alight detector. The scanning mirror device can be a linear galvanometer.The linear galvanometer can operate at a frequency of about 2 Hz toabout 120 Hz and a mechanical scan angle of about 10 degrees to about 20degrees. The laser can be a diode laser with a wavelength selected fromthe group consisting of 780 nm, 785 nm, 810 nm, and 830 nm.

Another embodiment of the invention provides a method for determining alocation of a resonant peak for a binding partner in a resonantreflectance spectrum with a colormetric resonant biosensor. The methodcomprises selecting a set of resonant reflectance data for a pluralityof colormetric resonant biosensor distinct locations. The set ofresonant reflectance data is collected by illuminating a colormetricresonant diffractive grating surface with a light source and measuringreflected light at a pre-determined incidence. The colormetric resonantdiffractive grating surface is used as a surface binding platform forone or more specific binding substances and binding partners can bedetected without the use of a molecular label. The set of resonantreflectance data includes a plurality of sets of two measurements, wherea first measurement includes a first reflectance spectra of one or morespecific binding substances that are attached to the colormetricresonant diffractive grating surface and a second measurement includes asecond reflectance spectra of the one or more specific bindingsubstances after one or more binding partners are applied to colormetricresonant diffractive grating surface including the one or more specificbinding substances. A difference in a peak wavelength between the firstand second measurement is a measurement of an amount of binding partnersthat bound to the one or more specific binding substances. A maximumvalue for a second measurement from the plurality of sets of twomeasurements from the set of resonant reflectance data for the pluralityof binding partners is determined, wherein the maximum value includesinherent noise included in the resonant reflectance data. Whether themaximum value is greater than a pre-determined threshold is determined,and if so, a curve-fit region around the determined maximum value isdetermined and a curve-fitting procedure is performed to fit a curvearound the curve-fit region, wherein the curve-fitting procedure removesa pre-determined amount of inherent noise included in the resonantreflectance data. A location of a maximum resonant peak on the fittedcurve is determined. A value of the maximum resonant peak is determined,wherein the value of the maximum resonant peak is used to identify anamount of biomolecular binding of the one or more specific bindingsubstances to the one or more binding partners.

The sensitivity of a colormetric resonant biosensor can be determined bya shift in a location of a resonant peak in the plurality of sets of twomeasurements in the set of resonant reflectance data. The step ofselecting a set of resonant reflectance data can include selecting a setof resonant reflectance data:x_(i) and y_(i) for i=1,2,3, . . . n,wherein x_(i) is where a first measurement includes a first reflectancespectra of one or more specific binding substance attached to thecolormetric resonant diffractive grating surface, y_(i) a secondmeasurement includes a second reflectance spectra of the one or morespecific binding substances after a plurality of binding partners areapplied to colormetric resonant diffractive grating surface includingthe one or more specific binding substances, and n is a total number ofmeasurements collected. The step of determining a maximum value for asecond measurement can include determining a maximum value y_(k) suchthat:(y _(k) >=y _(i)) for all i≠k.

The step of determining whether the maximum value is greater than apre-determined threshold can include computing a mean of the set ofresonant reflectance data, computing a standard deviation of the set ofresonant reflectance data, and determining whether((y_(k)−mean)/standard deviation) is greater than a pre-determinedthreshold. The step of defining a curve-fit region around the determinedmaximum value can include defining a curve-fit region of (2w+1) bins,wherein w is a pre-determined accuracy value, extracting (x_(i,),k−w<=i<=k+w), and extracting (y_(i), k−w<=i<=k+w). The step ofperforming a curve-fitting procedure can include computing g_(i)=lny_(i), performing a 2^(nd) order polynomial fit on g_(i) to obtaing′_(i) defined on (x_(i,), k−w<=i<=k+w), determining from the 2^(nd)order polynomial fit coefficients a, b and c of for (ax²+bx+c)−, andcomputing y′_(i)=e^(g′i). The step of determining a location of amaximum resonant peak on the fitted curve can include determininglocation of maximum resonant peak (x_(p)=(−b)/2a). The step ofdetermining a value of the maximum resonant peak can include determiningthe value with of x_(p) at y′_(p).

Even another embodiment of the invention comprises a computer readablemedium having stored therein instructions for causing a processor toexecute the methods for determining a location of a resonant peak for abinding partner in a resonant reflectance spectrum with a colormetricresonant biosensor, as described above.

Another embodiment of the invention provides a resonant reflectionstructure comprising a two-dimensional grating arranged in a pattern ofconcentric rings. The difference between an inside diameter and anoutside diameter of each concentric ring is equal to about one-half of agrating period, wherein each successive ring has an inside diameter thatis about one grating period greater than an inside diameter of aprevious ring. When the structure is illuminated with an illuminatinglight beam, a reflected radiation spectrum is produced that isindependent of an illumination polarization angle of the illuminatinglight beam. A resonant grating effect can be produced on the reflectedradiation spectrum, wherein the depth and period of the two-dimensionalgrating are less than the wavelength of the resonant grating effect, andwherein a narrow band of optical wavelengths is reflected from thestructure when the structure is illuminated with a broad band of opticalwavelengths. One or more specific binding substances can be immobilizedon the two-dimensional grating. The two-dimensional grating can have aperiod of about 0.01 microns to about 1 micron and a depth of about 0.1micron to about 1 micron.

Even another embodiment of the invention provides a transmission filterstructure comprising a two-dimensional grating arranged in a pattern ofconcentric rings. The difference between an inside diameter and anoutside diameter of each concentric ring is equal to about one-half of agrating period, wherein each successive ring has an inside diameter thatis about one grating period greater than an inside diameter of aprevious ring. When the structure is illuminated with an illuminatinglight beam, a transmitted radiation spectrum is produced that isindependent of an illumination polarization angle of the illuminatinglight beam. The structure of can be illuminated to produce a resonantgrating effect on the reflected radiation spectrum, wherein the depthand period of the two-dimensional grating are less than the wavelengthof the resonant grating effect, and wherein a narrow band of opticalwavelengths is reflected from the structure when the structure isilluminated with a broad band of optical wavelengths. One or morespecific binding substances can be immobilized on the two-dimensionalgrating. The two-dimensional grating can have a period of about 0.01microns to about 1 micron and a depth of about 0.01 microns to about 1micron.

Still another embodiment of the invention provides a resonant reflectionstructure comprising an array of holes or posts arranged such that theholes or posts are centered on the corners in the center of hexagons,wherein the hexagons are arranged in a closely packed array. When thestructure is illuminated with an illuminating light beam, a reflectedradiation spectrum is produced that is independent of an illuminationpolarization angle of the illuminating light beam. A resonant gratingeffect can be produced on the reflected radiation spectrum when thestructure is illuminated, wherein the depth or height and period of thearray of holes or posts are less than the wavelength of the resonantgrating effect, and wherein a narrow band of optical wavelengths isreflected from the structure when the structure is illuminated with abroad band of optical wavelengths. The resonant reflection structure canbe incorporated into a biosensor wherein one or more specific bindingsubstances are immobilized on the array of holes or posts. The holes orposts can have a period of about 0.01 microns to about 1 micron and adepth or height of about 0.01 microns to about 1 micron.

Yet another embodiment of the invention provides a transmission filterstructure comprising an array of holes or posts arranged such that theholes or posts are centered on the corners and in the center ofhexagons, wherein the hexagons are arranged in a closely packed array.When the structure is illuminated with an illuminating light beam, atransmitted radiation spectrum is produced that is independent of anillumination polarization angle of the illuminating light beam. When thestructure is illuminated a resonant grating effect is produced on thereflected radiation spectrum, wherein the depth or height and period ofthe array of holes or posts are less than the wavelength of the resonantgrating effect, and wherein a narrow band of optical wavelengths isreflected from the structure when the structure is illuminated with abroad band of optical wavelengths. The transmission filter structure canbe incorporated into a biosensor, wherein one or more specific bindingsubstances are immobilized on the array of holes or posts. The holes orposts can have a period of about 0.01 microns to about 1 micron and adepth or height of about 0.01 microns to about 1 micron.

Another embodiment of the invention provides a biosensor or opticaldevice comprising a first two-dimensional grating comprising a highrefractive index material and having a top surface and a bottom surface;and a second two-dimensional grating comprising a high refractive indexmaterial and having a top surface and a bottom surface, wherein the topsurface of the second two-dimensional grating is attached to the bottomsurface of the first two-dimensional grating. When the biosensor oroptical device is illuminated two resonant grating effects are producedon the reflected radiation spectrum and the depth and period of both ofthe two-dimensional gratings are less than the wavelength of theresonant grating effects. A substrate layer can support the bottomsurface of the second two-dimensional grating. The biosensor can furthercomprise one or more specific binding substances or one or more specificbinding substances bound to their binding partners immobilized on thetop surface of the first two-dimensional grating. The biosensor oroptical device can further comprising a cover layer on the top surfaceof the first two-dimensional grating, wherein the one or more specificbinding substances are immobilized on the surface of the cover layeropposite of the two-dimensional grating. The top surface of the firsttwo-dimensional grating can be in physical contact with a test sample,and the second two dimensional grating may not be in physical contactwith the test sample. A peak resonant reflection wavelength can bemeasured for the first and second two-dimensional gratings, thedifference between the two measurements indicates the amount of one ormore specific binding substances, binding partners or both deposited onthe surface of the first two-dimensional grating.

Even another embodiment of the invention provides a biosensor or opticaldevice comprising: a first two-dimensional grating comprising a highrefractive index material and having a top surface and a bottom surface,a substrate layer comprising a high refractive index material and havinga top surface and a bottom surface, wherein the top surface of thesubstrate supports the bottom surface of the first two-dimensionalgrating, and a second two-dimensional grating comprising a top surfaceand a bottom surface, wherein the bottom surface of the secondtwo-dimensional grating is attached to the bottom surface of thesubstrate. When the biosensor or optical device is illuminated tworesonant grating effects are produced on the reflected radiationspectrum, and wherein the depth and period of both of thetwo-dimensional gratings are less than the wavelength of the resonantgrating effects. The biosensor can comprise one or more specific bindingsubstances or one or more specific binding substances bound to theirbinding partners immobilized on the top surface of the firsttwo-dimensional grating. The biosensor or optical device can furthercomprise a cover layer on the top surface of the first two-dimensionalgrating, wherein the one or more specific binding substances areimmobilized on the surface of the cover layer opposite of thetwo-dimensional grating. The top surface of the first two-dimensionalgrating can be in physical contact with a test sample, and the secondtwo dimensional grating may not be in physical contact with the testsample. When a peak resonant reflection wavelength is measured for thefirst and second two-dimensional gratings, the difference between thetwo measurements can indicate the amount of one or more specific bindingsubstances, binding partners or both deposited on the surface of thefirst two-dimensional grating.

Still another embodiment of the invention provides a method of detectingan interaction of a first molecule with a second test molecule. Themethod comprises applying a mixture of the first and second molecules toa distinct location on a biosensor, wherein the biosensor comprises atwo-dimensional grating and a substrate layer that supports thetwo-dimensional grating; and wherein, when the biosensor is illuminateda resonant grating effect is produced on the reflected radiationspectrum, and wherein the depth and period of the two-dimensionalgrating are less than the wavelength of the resonant grating effect;applying a mixture of the first molecule with a third control moleculeto a distinct location on the biosensor or a similar biosensor, whereinthe third control molecule does not interact with the first molecule,and wherein the third control molecule is about the same size as thefirst molecule; and detecting a shift in the reflected wavelength oflight from the distinct locations. Wherein, if the shift in thereflected wavelength of light from the distinct location to which amixture of the first molecule and the second test molecule was appliedis greater than the shift in the reflected wavelength from the distinctlocation to which a mixture of the first molecule with the third controlmolecule was applied, then the first molecule and the second testmolecule interact. The first molecule can be selected from the groupconsisting of a nucleic acid, polypeptide, antigen, polyclonal antibody,monoclonal antibody, single chain antibody (scFv), F(ab) fragment,F(ab′)₂ fragment, Fv fragment, small organic molecule, cell, virus, andbacteria. The second test molecule can be selected from the groupconsisting of a nucleic acid, polypeptide, antigen, polyclonal antibody,monoclonal antibody, single chain antibody (scFv), F(ab) fragment,F(ab′)₂ fragment, Fv fragment, small organic molecule, cell, virus, andbacteria.

Therefore, unlike surface plasmon resonance, resonant mirrors, andwaveguide biosensors, the described compositions and methods enable manythousands of individual binding reactions to take place simultaneouslyupon the biosensor surface. This technology is useful in applicationswhere large numbers of biomolecular interactions are measured inparallel, particularly when molecular labels will alter or inhibit thefunctionality of the molecules under study. High-throughput screening ofpharmaceutical compound libraries with protein targets, and microarrayscreening of protein-protein interactions for proteomics are examples ofapplications that require the sensitivity and throughput afforded bythis approach. A biosensor of the invention can be manufactured, forexample, in large areas using a plastic embossing process, and thus canbe inexpensively incorporated into common disposable laboratory assayplatforms such as microtiter plates and microarray slides.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A-B show schematic diagrams of one embodiment of an opticalgrating structure used for a colormetric resonant reflectance biosensor.n_(substrate) represents substrate material. n₁ represents therefractive index of a cover layer. n₂ represents the refractive index ofa two-dimensional grating. n_(bio) represents the refractive index ofone or more specific binding substances. t₁ represents the thickness ofthe cover layer. t₂ represents the thickness of the grating. t_(bio)represents the thickness of the layer of one or more specific bindingsubstances. FIG. 1A shows a cross-sectional view of a biosensor. FIG. 1Bshows a diagram of a biosensor.

FIG. 2 shows a schematic drawing of a linear grating structure.

FIG. 3A-B shows a grating comprising a rectangular grid of squares (FIG.3A) or holes (FIG. 3B).

FIG. 4 shows a biosensor cross-section profile utilizing a sinusoidallyvarying grating profile.

FIG. 5 shows a biosensor cross-section profile in which an embossedsubstrate is coated with a higher refractive index material such as ZnSor SiN. A cover layer of, for example, epoxy or SOG is layered on top ofthe higher refractive index material and one or more specific bindingsubstances are immobilized on the cover layer.

FIG. 6 shows three types of surface activation chemistry (Amine,Aldehyde, and Nickel) with corresponding chemical linker molecules thatcan be used to covalently attach various types of biomolecule receptorsto a biosensor.

FIG. 7A-C shows methods that can be used to amplify the mass of abinding partner such as detected DNA or detected protein on the surfaceof a biosensor.

FIG. 8 shows a graphic representation of how adsorbed material, such asa protein monolayer, will increase the reflected wavelength of on a SRVDbiosensor.

FIG. 9 shows an example of a biosensor used as a microarray.

FIG. 10A-B shows two biosensor formats that can incorporate acolorimetric resonant reflectance biosensor. FIG. 10A shows a biosensorthat is incorporated into a microtitre plate. FIG. 10B shows a biosensorin a microarray slide format.

FIG. 11 shows an array of arrays concept for using a biosensor platformto perform assays with higher density and throughput.

FIG. 12 shows a diagram of an array of biosensor electrodes. A singleelectrode can comprise a region that contains many grating periods andseveral separate grating regions can occur on the same substratesurface.

FIG. 13 shows a SEM photograph showing the separate grating regions ofan array of biosensor electrodes.

FIG. 14 shows a biosensor upper surface immersed in a liquid sample. Anelectrical potential can be applied to the biosensor that is capable ofattracting or repelling a biomolecule near the electrode surface.

FIG. 15 shows a biosensor upper surface immersed in a liquid sample. Apositive voltage is applied to an electrode and the electronegativebiomolecules are attracted to the biosensor surface.

FIG. 16 shows a biosensor upper surface immersed in a liquid sample. Anegative voltage is applied to an electrode and the electronegativebiomolecules are repelled from the biosensor surface using a negativeelectrode voltage.

FIG. 17 demonstrates an example of a biosensor that occurs on the tip ofa fiber probe for in vivo detection of biochemical substances.

FIG. 18 shows an example of the use of two coupled fibers to illuminateand collect reflected light from a biosensor.

FIG. 19 shows resonance wavelength of a biosensor as a function ofincident angle of detection beam.

FIG. 20 shows an example of the use of a beam splitter to enableilluminating and reflected light to share a common collimated opticalpath to a biosensor.

FIG. 21 shows an example of a system for angular scanning of abiosensor.

FIG. 22 shows SEM photographs of a photoresist grating structure in planview (center and upper right) and cross-section (lower right).

FIG. 23 shows a SEM cross-section photograph of a grating structureafter spin-on glass is applied over a silicon nitride grating.

FIG. 24 shows examples of biosensor chips (1.5×1.5-inch). Circular areasare regions where the resonant structure is defined.

FIG. 25 shows response as a function of wavelength of a biosensor thatBSA had been deposited at high concentration, measured in air. Beforeprotein deposition, the resonant wavelength of the biosensor is 380 nmand is not observable with the instrument used for this experiment.

FIG. 26 shows response as a function of wavelength comparing anuntreated biosensor with one upon which BSA had been deposited. Bothmeasurements were taken with water on the biosensor's surface.

FIG. 27 shows response as a function of wavelength of a biosensor thatBorrelia bacteria has been deposited at high concentration and measuredin water.

FIG. 28 shows a computer simulation of a biosensor demonstrating theshift of resonance to longer wavelengths as biomolecules are depositedon the surface.

FIG. 29 shows a computer simulation demonstrating the dependence of peakreflected wavelength on protein coating thickness. This particularbiosensor has a dynamic range of 250 nm deposited biomaterial before theresponse begins to saturate.

FIG. 30 shows an embodiment of a biosensor. n_(substrate) represents therefractive index of a substrate. n₁ represents the refractive index ofan optical cover layer. n₂ represents the refractive index of atwo-dimensional grating. n₃ represents the refractive index of a highrefractive index material such as silicon nitride. n_(bio) representsthe refractive index of one or more specific binding substances. t₁represents the thickness of a cover layer. t₂ represents the thicknessof a two-dimensional grating. t₃ represents the thickness of a highrefractive index material. t_(bio) represents the thickness of aspecific binding substance layer.

FIG. 31 shows reflected intensity as a function of wavelength for aresonant grating structure when various thicknesses of protein areincorporated onto the upper surface.

FIG. 32 shows a linear relationship between reflected wavelength andprotein coating thickness for a biosensor shown in FIG. 30.

FIG. 33 shows instrumentation that can be used to read output of abiosensor. A collimated light source is directed at a biosensor surfaceat normal incidence through an optical fiber, while a second parallelfiber collects the light reflected at normal incidence. A spectrometerrecords the reflectance as a function of wavelength.

FIG. 34 shows the measured reflectance spectra of a biosensor.

FIG. 35 shows dependence of peak resonant wavelength measured in liquidupon the concentration of protein BSA dissolved in water.

FIG. 36 shows dependence of peak resonance wavelength on theconcentration of BSA dissolved in PBS, which was then allowed to dry ona biosensor surface.

FIG. 37A-B. FIG. 37A shows a measurement of peak resonant wavelengthshift caused by attachment of a streptavidin receptor layer andsubsequent detection of a biotinylated IgG. FIG. 37B shows a schematicdemonstration of molecules bound to a biosensor.

FIG. 38A-B. FIG. 38A shows results of streptavidin detection at variousconcentrations for a biosensor that has been activated with NH₂ surfacechemistry linked to a biotin receptor molecule. FIG. 38B shows aschematic demonstration of molecules bound to a biosensor.

FIG. 39A-B. FIG. 39A shows an assay for detection of anti-goat IgG usinga goat antibody receptor molecule. BSA blocking of a detection surfaceyields a clearly measurable background signal due to the mass of BSAincorporated on the biosensor. A 66 nM concentration of anti-goat IgG iseasily measured above the background signal. FIG. 39B shows a schematicdemonstration of molecules bound to a biosensor.

FIG. 40A-B. FIG. 40A shows a nonlabeled ELISA assay for interferon-gamma(INF-gamma) using an anti-human IgG INF-gamma receptor molecule, and aneural growth factor (NGF) negative control. FIG. 40B shows a schematicdemonstration of molecules bound to a biosensor.

FIG. 41A-B. FIG. 41A shows detection of a 5-amino acid peptide (MW=860)and subsequent cleavage of a pNA label (MW=130) using enzyme caspase-3.FIG. 41B shows a schematic demonstration of molecules bound to abiosensor.

FIG. 42A-B. FIG. 42A shows resonant peak in liquid during continuousmonitoring of the binding of three separate protein layers. FIG. 42Bshows a schematic demonstration of molecules bound to a biosensor.

FIG. 43A-B. FIG. 43A shows endpoint resonant frequencies mathematicallydetermined from the data shown in FIG. 42. FIG. 43B shows a schematicdemonstration of molecules bound to a biosensor.

FIG. 44A-B. FIG. 44A shows kinetic binding measurement of IgG binding.FIG. 44B shows a schematic demonstration of molecules bound to abiosensor.

FIG. 45A-B. FIG. 45A shows kinetic measurement of a protease thatcleaves bound protein from a biosensor surface. FIG. 45B shows aschematic demonstration of molecules bound to a biosensor.

FIG. 46 shows comparison of mathematical fit of parabolic andexponential functions to spectrometer data from a resonant peak. Theexponential curve fit is used to mathematically determine a peakresonant wavelength.

FIG. 47 shows sensitivity of the mathematically determined peak resonantwavelength to artificially added noise in the measured spectrum.

FIG. 48 shows a resonant optical biosensor incorporating an electricallyconducting material.

FIG. 49 shows a resonant reflection or transmission filter structureconsisting of a set of concentric rings.

FIG. 50 shows a resonant reflective or transmission filter structurecomprising a hexagonal grid of holes (or a hexagonal grid of posts) thatclosely approximates the concentric circle structure of FIG. 49 withoutrequiring the illumination beam to be centered upon any particularlocation of the grid.

FIG. 51 shows a plot of the peak resonant wavelength values for testsolutions. The avidin solution was taken as the baseline reference forcomparison to the Avidin+BSA and Avidin+b-BSA solutions. Addition of BSAto avidin results in only a small resonant wavelength increase, as thetwo proteins are not expected to interact. However, because biotin andavidin bind strongly (Kd=10⁻¹⁵M), the avidin+b-BSA solution will containlarger bound protein complexes. The peak resonant wavelength value ofthe avidin+b-BSA solution thus provides a large shift compared toavidin+BSA.

FIG. 52 shows a schematic diagram of a detection system.

DETAILED DESCRIPTION OF THE INVENTION

Subwavelength Structured Surface (SWS) Biosensor

In one embodiment of the invention, a subwavelength structured surface(SWS) is used to create a sharp optical resonant reflection at aparticular wavelength that can be used to track with high sensitivitythe interaction of biological materials, such as specific bindingsubstances or binding partners or both. A colormetric resonantdiffractive grating surface acts as a surface binding platform forspecific binding substances.

Subwavelength structured surfaces are an unconventional type ofdiffractive optic that can mimic the effect of thin-film coatings. (Peng& Morris, “Resonant scattering from two-dimensional gratings,” J. Opt.Soc. Am. A, Vol. 13, No. 5, p. 993, May; Magnusson, & Wang, “Newprinciple for optical filters,” Appl. Phys. Lett., 61, No. 9, p. 1022,August, 1992; Peng & Morris, “Experimental demonstration of resonantanomalies in diffraction from two-dimensional gratings,” Optics Letters,Vol. 21, No. 8, p. 549, April, 1996). A SWS structure contains asurface-relief, two-dimensional grating in which the grating period issmall compared to the wavelength of incident light so that nodiffractive orders other than the reflected and transmitted zerothorders are allowed to propagate. A SWS surface narrowband filter cancomprise a two-dimensional grating sandwiched between a substrate layerand a cover layer that fills the grating grooves. Optionally, a coverlayer is not used. A resonant grating effect can be produced on thereflected radiation spectrum, resulting in a narrow band of reflected ortransmitted wavelengths. The depth and period of the two-dimensionalgrating are less than the wavelength of the resonant grating effect.

The reflected or transmitted color of this structure can be modulated bythe addition of molecules such as specific binding substances or bindingpartners or both to the upper surface of the cover layer or thetwo-dimensional grating surface. The added molecules increase theoptical path length of incident radiation through the structure, andthus modify the wavelength at which maximum reflectance or transmittancewill occur.

In one embodiment, a biosensor, when illuminated with white light, isdesigned to reflect only a single wavelength. When specific bindingsubstances are attached to the surface of the biosensor, the reflectedwavelength (color) is shifted due to the change of the optical path oflight that is coupled into the grating. By linking specific bindingsubstances to a biosensor surface, complementary binding partnermolecules can be detected without the use of any kind of fluorescentprobe or particle label. The detection technique is capable of resolvingchanges of, for example, ˜0.1 nm thickness of protein binding, and canbe performed with the biosensor surface either immersed in fluid ordried.

A detection system consists of, for example, a light source thatilluminates a small spot of a biosensor at normal incidence through, forexample, a fiber optic probe, and a spectrometer that collects thereflected light through, for example, a second fiber optic probe also atnormal incidence. Because no physical contact occurs between theexcitation/detection system and the biosensor surface, no specialcoupling prisms are required and the biosensor can be easily adapted toany commonly used assay platform including, for example, microtiterplates and microarray slides. A single spectrometer reading can beperformed in several milliseconds, thus it is possible to quicklymeasure a large number of molecular interactions taking place inparallel upon a biosensor surface, and to monitor reaction kinetics inreal time.

This technology is useful in applications where large numbers ofbiomolecular interactions are measured in parallel, particularly whenmolecular labels would alter or inhibit the functionality of themolecules under study. High-throughput screening of pharmaceuticalcompound libraries with protein targets, and microarray screening ofprotein-protein interactions for proteomics are examples of applicationsthat require the sensitivity and throughput afforded by the compositionsand methods of the invention.

A schematic diagram of an example of a SWS structure is shown in FIG. 1.In FIG. 1, n_(substrate) represents a substrate material. n₁ representsthe refractive index of an optional cover layer. n₂ represents therefractive index of a two-dimensional grating. N_(bio) represents therefractive index of one or more specific binding substances. t₁represents the thickness of the cover layer above the two-dimensionalgrating structure. t₂ represents the thickness of the grating. t_(bio)represents the thickness of the layer of one or more specific bindingsubstances. In one embodiment, are n2<n1 (see FIG. 1). Layer thicknesses(i.e. cover layer, one or more specific binding substances, or atwo-dimensional grating) are selected to achieve resonant wavelengthsensitivity to additional molecules on the top surface The gratingperiod is selected to achieve resonance at a desired wavelength.

One embodiment of the invention provides a SWS biosensor. A SWSbiosensor comprises a two-dimensional grating, a substrate layer thatsupports the two-dimensional grating, and one or more specific bindingsubstances immobilized on the surface of the two-dimensional gratingopposite of the substrate layer.

A two-dimensional grating can be comprised of a material, including, forexample, zinc sulfide, titanium dioxide, tantalum oxide, and siliconnitride. A cross-sectional profile of a two-dimensional grating cancomprise any periodically repeating function, for example, a“square-wave.” A two-dimensional grating can be comprised of a repeatingpattern of shapes selected from the group consisting of lines, squares,circles, ellipses, triangles, trapezoids, sinusoidal waves, ovals,rectangles, and hexagons. A sinusoidal cross-sectional profile ispreferable for manufacturing applications that require embossing of agrating shape into a soft material such as plastic. In one embodiment ofthe invention, the depth of the grating is about 0.01 micron to about 1micron and the period of the grating is about 0.01 micron to about 1micron.

Linear gratings have resonant characteristics where the illuminatinglight polarization is oriented perpendicular to the grating period.However, a hexagonal grid of holes has better polarization symmetry thana rectangular grid of holes. Therefore, a colorimetric resonantreflection biosensor of the invention can comprise, for example, ahexagonal array of holes (see FIG. 3B) or a grid of parallel lines (seeFIG. 3A). A linear grating has the same pitch (i.e. distance betweenregions of high and low refractive index), period, layer thicknesses,and material properties as the hexagonal array grating. However, lightmust be polarized perpendicular to the grating lines in order to beresonantly coupled into the optical structure. Therefore, a polarizingfilter oriented with its polarization axis perpendicular to the lineargrating must be inserted between the illumination source and thebiosensor surface. Because only a small portion of the illuminatinglight source is correctly polarized, a longer integration time isrequired to collect an equivalent amount of resonantly reflected lightcompared to a hexagonal grating.

While a linear grating can require either a higher intensityillumination source or a longer measurement integration time compared toa hexagonal grating, the fabrication requirements for the linearstructure are simpler. A hexagonal grating pattern is produced byholographic exposure of photoresist to three mutually interfering laserbeams. The three beams are precisely aligned in order to produce agrating pattern that is symmetrical in three directions. A lineargrating pattern requires alignment of only two laser beams to produce aholographic exposure in photoresist, and thus has a reduced alignmentrequirement. A linear grating pattern can also be produced by, forexample, direct writing of photoresist with an electron beam. Also,several commercially available sources exist for producing lineargrating “master” templates for embossing a grating structure intoplastic. A schematic diagram of a linear grating structure is shown inFIG. 2.

A rectangular grid pattern can be produced in photoresist using anelectron beam direct-write exposure system. A single wafer can beilluminated as a linear grating with two sequential exposures with thepart rotated 90-degrees between exposures.

A two-dimensional grating can also comprise, for example, a “stepped”profile, in which high refractive index regions of a single, fixedheight are embedded within a lower refractive index cover layer. Thealternating regions of high and low refractive index provide an opticalwaveguide parallel to the top surface of the biosensor. See FIG. 5.

For manufacture, a stepped structure is etched or embossed into asubstrate material such as glass or plastic. See FIG. 5. A uniform thinfilm of higher refractive index material, such as silicon nitride orzinc sulfide is deposited on this structure. The deposited layer willfollow the shape contour of the embossed or etched structure in thesubstrate, so that the deposited material has a surface relief profilethat is identical to the original embossed or etched profile. Thestructure can be completed by the application of an optional cover layercomprised of a material having a lower refractive index than the higherrefractive index material and having a substantially flat upper surface.The covering material can be, for example, glass, epoxy, or plastic.

This structure allows for low cost biosensor manufacturing, because itcan be mass produced. A “master” grating can be produced in glass,plastic, or metal using, for example, a three-beam laser holographicpatterning process, See e.g., Cowan, The recording and large scaleproduction of crossed holographic grating arrays using multiple beaminterferometry, Proc. Soc. Photo-optical Instum. Eng. 503:120 (1984). Amaster grating can be repeatedly used to emboss a plastic substrate. Theembossed substrate is subsequently coated with a high refractive indexmaterial and optionally, a cover layer.

While a stepped structure is simple to manufacture, it is also possibleto make a resonant biosensor in which the high refractive index materialis not stepped, but which varies with lateral position. FIG. 4 shows aprofile in which the high refractive index material of thetwo-dimensional grating, n₂, is sinusoidally varying in height. Toproduce a resonant reflection at a particular wavelength, the period ofthe sinusoid is identical to the period of an equivalent steppedstructure. The resonant operation of the sinusoidally varying structureand its functionality as a biosensor has been verified using GSOLVER(Grating Solver Development Company, Allen, Tex., USA) computer models.

Techniques for making two-dimensional gratings are disclosed in Wang, J.Opt. Soc. Am No. 8, August 1990, pp. 1529-44. Biosensors of theinvention can be made in, for example, a semiconductor microfabricationfacility. Biosensors can also be made on a plastic substrate usingcontinuous embossing and optical coating processes. For this type ofmanufacturing process, a “master” structure is built in a rigid materialsuch as glass or silicon, and is used to generate “mother” structures inan epoxy or plastic using one of several types of replicationprocedures. The “mother” structure, in turn, is coated with a thin filmof conducive material, and used as a mold to electroplate a thick filmof nickel. The nickel “daughter” is released from the plastic “mother”structure. Finally, the nickel “daughter” is bonded to a cylindricaldrum, which is used to continuously emboss the surface relief structureinto a plastic film. A device structure that uses an embossed plasticsubstrate is shown in FIG. 5. Following embossing, the plastic structureis overcoated with a thin film of high refractive index material, andoptionally coated with a planarizing, cover layer polymer, and cut toappropriate size.

A substrate for a SWS biosensor can comprise, for example, glass,plastic or epoxy. Optionally, a substrate and a two-dimensional gratingcan comprise a single unit. That is, a two dimensional grating andsubstrate are formed from the same material, for example, glass,plastic, or epoxy. The surface of a single unit comprising thetwo-dimensional grating is coated with a material having a highrefractive index, for example, zinc sulfide, titanium dioxide, tantalumoxide, and silicon nitride. One or more specific binding substances canbe immobilized on the surface of the material having a high refractiveindex or on an optional cover layer.

A biosensor of the invention can further comprise a cover layer on thesurface of a two-dimensional grating opposite of a substrate layer.Where a cover layer is present, the one or more specific bindingsubstances are immobilized on the surface of the cover layer opposite ofthe two-dimensional grating. Preferably, a cover layer comprises amaterial that has a lower refractive index than a material thatcomprises the two-dimensional grating. A cover layer can be comprisedof, for example, glass (including spin-on glass (SOG)), epoxy, orplastic.

For example, various polymers that meet the refractive index requirementof a biosensor can be used for a cover layer. SOG can be used due to itsfavorable refractive index, ease of handling, and readiness of beingactivated with specific binding substances using the wealth of glasssurface activation techniques. When the flatness of the biosensorsurface is not an issue for a particular system setup, a gratingstructure of SiN/glass can directly be used as the sensing surface, theactivation of which can be done using the same means as on a glasssurface.

Resonant reflection can also be obtained without a planarizing coverlayer over a two-dimensional grating. For example, a biosensor cancontain only a substrate coated with a structured thin film layer ofhigh refractive index material. Without the use of a planarizing coverlayer, the surrounding medium (such as air or water) fills the grating.Therefore, specific binding substances are immobilized to the biosensoron all surfaces a two-dimensional grating exposed to the specificbinding substances, rather than only on an upper surface.

In general, a biosensor of the invention will be illuminated with whitelight that will contain light of every polarization angle. Theorientation of the polarization angle with respect to repeating featuresin a biosensor grating will determine the resonance wavelength. Forexample, a “linear grating” biosensor structure consisting of a set ofrepeating lines and spaces will have two optical polarizations that cangenerate separate resonant reflections. Light that is polarizedperpendicularly to the lines is called “s-polarized,” while light thatis polarized parallel to the lines is called “p-polarized.” Both the sand p components of incident light exist simultaneously in an unfilteredillumination beam, and each generates a separate resonant signal. Abiosensor structure can generally be designed to optimize the propertiesof only one polarization (the s-polarization), and the non-optimizedpolarization is easily removed by a polarizing filter.

In order to remove the polarization dependence, so that everypolarization angle generates the same resonant reflection spectra, analternate biosensor structure can be used that consists of a set ofconcentric rings. In this structure, the difference between the insidediameter and the outside diameter of each concentric ring is equal toabout one-half of a grating period. Each successive ring has an insidediameter that is about one grating period greater than the insidediameter of the previous ring. The concentric ring pattern extends tocover a single sensor location—such as a microarray spot or a microtiterplate well. Each separate microarray spot or microtiter plate well has aseparate concentric ring pattern centered within it. e.g., FIG. 49. Allpolarization directions of such a structure have the samecross-sectional profile. The concentric ring structure must beilluminated precisely on-center to preserve polarization independence.The grating period of a concentric ring structure is less than thewavelength of the resonantly reflected light. The grating period isabout 0.01 micron to about 1 micron. The grating depth is about 0.01 toabout 1 micron.

In another embodiment, an array of holes or posts are arranged toclosely approximate the concentric circle structure described abovewithout requiring the illumination beam to be centered upon anyparticular location of the grid. See e.g. FIG. 50. Such an array patternis automatically generated by the optical interference of three laserbeams incident on a surface from three directions at equal angles. Inthis pattern, the holes (or posts) are centered upon the corners of anarray of closely packed hexagons as shown in FIG. 50. The holes or postsalso occur in the center of each hexagon. Such a hexagonal grid of holesor posts has three polarization directions that “see” the samecross-sectional profile. The hexagonal grid structure, therefore,provides equivalent resonant reflection spectra using light of anypolarization angle. Thus, no polarizing filter is required to removeunwanted reflected signal components. The period of the holes or postscan be about 0.01 microns to about 1 micron and the depth or height canbe about 0.01 microns to about 1 micron.

The invention provides a resonant reflection structures and transmissionfilter structures comprising concentric circle gratings and hexagonalgrids of holes or posts. For a resonant reflection structure, lightoutput is measured on the same side of the structure as the illuminatinglight beam. For a transmission filter structure, light output ismeasured on the opposite side of the structure as the illuminating beam.The reflected and transmitted signals are complementary. That is, if awavelength is strongly reflected, it is weakly transmitted. Assuming noenergy is absorbed in the structure itself, the reflected+transmittedenergy at any given wavelength is constant. The resonant reflectionstructure and transmission filters are designed to give a highlyefficient reflection at a specified wavelength. Thus, a reflectionfilter will “pass” a narrow band of wavelengths, while a transmissionfilter will “cut” a narrow band of wavelengths from incident light.

A resonant reflection structure or a transmission filter structure cancomprise a two-dimensional grating arranged in a pattern of concentriccircles. A resonant reflection structure or transmission filterstructure can also comprise a hexagonal grid of holes or posts. Whenthese structure are illuminated with an illuminating light beam, areflected radiation spectrum is produced that is independent of anillumination polarization angle of the illuminating light beam. Whenthese structures are illuminated a resonant grating effect is producedon the reflected radiation spectrum, wherein the depth and period of thetwo-dimensional grating or hexagonal grid of holes or posts are lessthan the wavelength of the resonant grating effect. These structuresreflect a narrow band of light when the structure is illuminated with abroadband of light.

Resonant reflection structures and transmission filter structures of theinvention can be used as biosensors. For example, one or more specificbinding substances can be immobilized on the hexagonal grid of holes orposts or on the two-dimensional grating arranged in concentric circles.

In one embodiment of the invention, a reference resonant signal isprovided for more accurate measurement of peak resonant wavelengthshifts. The reference resonant signal can cancel out environmentaleffects, including, for example, temperature. A reference signal can beprovided using a resonant reflection superstructure that produces twoseparate resonant wavelengths. A transparent resonant reflectionsuperstructure can contain two sub-structures. A first sub-structurecomprises a first two-dimensional grating with a top and a bottomsurface. The top surface of a two-dimensional grating comprises thegrating surface. The first two-dimensional grating can comprise one ormore specific binding substances immobilized on its top surface. The topsurface of the first two-dimensional grating is in contact with a testsample. An optional substrate layer can be present to support the bottomsurface of the first two-dimensional grating. The substrate layercomprises a top and bottom surface. The top surface of the substrate isin contact with, and supports the bottom surface of the firsttwo-dimensional grating.

A second sub-structure comprises a second two-dimensional grating with atop surface and a bottom surface. The second two-dimensional grating isnot in contact with a test sample. The second two-dimensional gratingcan be fabricated onto the bottom surface of the substrate that supportsthe first two-dimensional grating. Where the second two-dimensionalgrating is fabricated on the substrate that supports the firsttwo-dimensional grating, the bottom surface of the secondtwo-dimensional grating can be fabricated onto the bottom surface of thesubstrate. Therefore, the top surface of the second two-dimensionalgrating will face the opposite direction of the top surface of the firsttwo-dimensional grating.

The top surface of the second two-dimensional grating can also beattached directly to the bottom surface of the first sub-structure. Inthis embodiment the top surface of the second two-dimensional gratingwill face the same direction as the top surface of the firsttwo-dimensional grating. A substrate can support the bottom surface ofthe second two-dimensional grating in this embodiment.

Because the second sub-structure is not in physical contact with thetest sample, its peak resonant wavelength is not subject to changes inthe optical density of the test media, or deposition of specific bindingsubstances or binding partners on the surface of the firsttwo-dimensional grating. Therefore, such a superstructure produces tworesonant signals. Because the location of the peak resonant wavelengthin the second sub-structure is fixed, the difference in peak resonantwavelength between the two sub-structures provides a relative means fordetermining the amount of specific binding substances or bindingpartners or both deposited on the top surface of the first substructurethat is exposed to the test sample.

A biosensor superstructure can be illuminated from its top surface orfrom its bottom surface, or from both surfaces. The peak resonancereflection wavelength of the first substructure is dependent on theoptical density of material in contact with the superstructure surface,while the peak resonance reflection wavelength of the secondsubstructure is independent of the optical density of material incontact with the superstructure surface.

In one embodiment of the invention, a biosensor is illuminated from thebottom surface of the biosensor. Approximately 50% of the incident lightis reflected from the bottom surface of biosensor without reaching theactive (top) surface of the biosensor. A thin film or physical structurecan be included in a biosensor composition that is capable of maximizingthe amount of light that is transmitted to the upper surface of thebiosensor while minimizing the reflected energy at the resonantwavelength. The anti-reflection thin film or physical structure of thebottom surface of the biosensor can comprise, for example, a singledielectric thin film, a stack of multiple dielectric thin films, or a“motheye” structure that is embossed into the bottom biosensor surface.An example of a motheye structure is disclosed in Hobbs, et al.“Automated interference lithography system for generation of sub-micronfeature size patterns,” Proc. 1999 Micromachine Technology forDiffracting and Holographic Optics, Society of Photo-OpticalInstrumentation Engineers, p. 124-135, (1999).

In one embodiment of the invention, an optical device is provided. Anoptical device comprises a structure similar to any biosensor of theinvention; however, an optical device does not comprise one of morebinding substances immobilized on the two-dimensional grating. Anoptical device can be used as a narrow band optical filter.

In one embodiment of the invention, an interaction of a first moleculewith a second test molecule can be detected. A SWS biosensor asdescribed above is used; however, there are no specific bindingsubstances immobilized on its surface. Therefore, the biosensorcomprises a two-dimensional grating, a substrate layer that supports thetwo-dimensional grating, and optionally, a cover layer. As describedabove, when the biosensor is illuminated a resonant grating effect isproduced on the reflected radiation spectrum, and the depth and periodof the two-dimensional grating are less than the wavelength of theresonant grating effect.

To detect an interaction of a first molecule with a second testmolecule, a mixture of the first and second molecules is applied to adistinct location on a biosensor. A distinct location can be one spot orwell on a biosensor or can be a large area on a biosensor. A mixture ofthe first molecule with a third control molecule is also applied to adistinct location on a biosensor. The biosensor can be the samebiosensor as described above, or can be a second biosensor. If thebiosensor is the same biosensor, a second distinct location can be usedfor the mixture of the first molecule and the third control molecule.Alternatively, the same distinct biosensor location can be used afterthe first and second molecules are washed from the biosensor. The thirdcontrol molecule does not interact with the first molecule and is aboutthe same size as the first molecule. A shift in the reflected wavelengthof light from the distinct locations of the biosensor or biosensors ismeasured. If the shift in the reflected wavelength of light from thedistinct location having the first molecule and the second test moleculeis greater than the shift in the reflected wavelength from the distinctlocation having the first molecule and the third control molecule, thenthe first molecule and the second test molecule interact. Interactioncan be, for example, hybridization of nucleic acid molecules, specificbinding of an antibody or antibody fragment to an antigen, and bindingof polypeptides. A first molecule, second test molecule, or thirdcontrol molecule can be, for example, a nucleic acid, polypeptide,antigen, polyclonal antibody, monoclonal antibody, single chain antibody(scFv), F(ab) fragment, F(ab′)₂ fragment, Fv fragment, small organicmolecule, cell, virus, and bacteria.

Specific Binding Substances and Binding Partners

One or more specific binding substances are immobilized on thetwo-dimensional grating or cover layer, if present, by for example,physical adsorption or by chemical binding. A specific binding substancecan be, for example, a nucleic acid, polypeptide, antigen, polyclonalantibody, monoclonal antibody, single chain antibody (scFv), F(ab)fragment, F(ab′)₂ fragment, Fv fragment, small organic molecule, cell,virus, bacteria, or biological sample. A biological sample can be forexample, blood, plasma, serum, gastrointestinal secretions, homogenatesof tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid,amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavagefluid, semen, lymphatic fluid, tears, or prostatitc fluid.

Preferably, one or more specific binding substances are arranged in amicroarray of distinct locations on a biosensor. A microarray ofspecific binding substances comprises one or more specific bindingsubstances on a surface of a biosensor of the invention such that asurface contains many distinct locations, each with a different specificbinding substance or with a different amount of a specific bindingsubstance. For example, an array can comprise 1, 10, 100, 1,000, 10,000,or 100,000 distinct locations. Such a biosensor surface is called amicroarray because one or more specific binding substances are typicallylaid out in a regular grid pattern in x-y coordinates. However, amicroarray of the invention can comprise one or more specific bindingsubstance laid out in any type of regular or irregular pattern. Forexample, distinct locations can define a microarray of spots of one ormore specific binding substances. A microarray spot can be about 50 toabout 500 microns in diameter. A microarray spot can also be about 150to about 200 microns in diameter. One or more specific bindingsubstances can be bound to their specific binding partners.

A microarray on a biosensor of the invention can be created by placingmicrodroplets of one or more specific binding substances onto, forexample, an x-y grid of locations on a two-dimensional grating or coverlayer surface. When the biosensor is exposed to a test sample comprisingone or more binding partners, the binding partners will bepreferentially attracted to distinct locations on the microarray thatcomprise specific binding substances that have high affinity for thebinding partners. Some of the distinct locations will gather bindingpartners onto their surface, while other locations will not.

A specific binding substance specifically binds to a binding partnerthat is added to the surface of a biosensor of the invention. A specificbinding substance specifically binds to its binding partner, but doesnot substantially bind other binding partners added to the surface of abiosensor. For example, where the specific binding substance is anantibody and its binding partner is a particular antigen, the antibodyspecifically binds to the particular antigen, but does not substantiallybind other antigens. A binding partner can be, for example, a nucleicacid, polypeptide, antigen, polyclonal antibody, monoclonal antibody,single chain antibody (scFv), F(ab) fragment, F(ab′)₂ fragment, Fvfragment, small organic molecule, cell, virus, bacteria, and biologicalsample. A biological sample can be, for example, blood, plasma, serum,gastrointestinal secretions, homogenates of tissues or tumors, synovialfluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinalfluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid,tears, and prostatitc fluid.

One example of a microarray of the invention is a nucleic acidmicroarray, in which each distinct location within the array contains adifferent nucleic acid molecule. In this embodiment, the spots withinthe nucleic acid microarray detect complementary chemical binding withan opposing strand of a nucleic acid in a test sample.

While microtiter plates are the most common format used for biochemicalassays, microarrays are increasingly seen as a means for maximizing thenumber of biochemical interactions that can be measured at one timewhile minimizing the volume of precious reagents. By application ofspecific binding substances with a microarray spotter onto a biosensorof the invention, specific binding substance densities of 10,000specific binding substances/in² can be obtained. By focusing anillumination beam to interrogate a single microarray location, abiosensor can be used as a label-free microarray readout system.

Immobilization or One or More Specific Binding Substances

Immobilization of one or more binding substances onto a biosensor isperformed so that a specific binding substance will not be washed awayby rinsing procedures, and so that its binding to binding partners in atest sample is unimpeded by the biosensor surface. Several differenttypes of surface chemistry strategies have been implemented for covalentattachment of specific binding substances to, for example, glass for usein various types of microarrays and biosensors. These same methods canbe readily adapted to a biosensor of the invention. Surface preparationof a biosensor so that it contains the correct functional groups forbinding one or more specific binding substances is an integral part ofthe biosensor manufacturing process.

One or more specific binding substances can be attached to a biosensorsurface by physical adsorption (i.e., without the use of chemicallinkers) or by chemical binding (i.e., with the use of chemicallinkers). Chemical binding can generate stronger attachment of specificbinding substances on a biosensor surface and provide definedorientation and conformation of the surface-bound molecules.

Several examples of chemical binding of specific binding substances to abiosensor of the invention appear in Example 8, below. Other types ofchemical binding include, for example, amine activation, aldehydeactivation, and nickel activation. These surfaces can be used to attachseveral different types of chemical linkers to a biosensor surface, asshown in FIG. 6. While an amine surface can be used to attach severaltypes of linker molecules, an aldehyde surface can be used to bindproteins directly, without an additional linker. A nickel surface can beused to bind molecules that have an incorporated histidine (“his”) tag.Detection of “his-tagged” molecules with a nickel-activated surface iswell known in the art (Whitesides, Anal. Chem. 68, 490, (1996)).

Immobilization of specific binding substances to plastic, epoxy, or highrefractive index material can be performed essentially as described forimmobilization to glass. However, the acid wash step can be eliminatedwhere such a treatment would damage the material to which the specificbinding substances are immobilized.

For the detection of binding partners at concentrations less than about˜0.1 ng/ml, it is preferable to amplify and transduce binding partnersbound to a biosensor into an additional layer on the biosensor surface.The increased mass deposited on the biosensor can be easily detected asa consequence of increased optical path length. By incorporating greatermass onto a biosensor surface, the optical density of binding partnerson the surface is also increased, thus rendering a greater resonantwavelength shift than would occur without the added mass. The additionof mass can be accomplished, for example, enzymatically, through a“sandwich” assay, or by direct application of mass to the biosensorsurface in the form of appropriately conjugated beads or polymers ofvarious size and composition. This principle has been exploited forother types of optical biosensors to demonstrate sensitivity increasesover 1500× beyond sensitivity limits achieved without massamplification. See, e.g., Jenison et al., “Interference-based detectionof nucleic acid targets on optically coated silicon,” NatureBiotechnology, 19: 62-65, 2001.

As an example, FIG. 7A shows that an NH₂-activated biosensor surface canhave a specific binding substance comprising a single-strand DNA captureprobe immobilized on the surface. The capture probe interactsselectively with its complementary target binding partner. The bindingpartner, in turn, can be designed to include a sequence or tag that willbind a “detector” molecule. As shown in FIG. 7A, a detector molecule cancontain, for example, a linker to horseradish peroxidase (HRP) that,when exposed to the correct enzyme, will selectively deposit additionalmaterial on the biosensor only where the detector molecule is present.Such a procedure can add, for example, 300 angstroms of detectablebiomaterial to the biosensor within a few minutes.

A “sandwich” approach can also be used to enhance detection sensitivity.In this approach, a large molecular weight molecule can be used toamplify the presence of a low molecular weight molecule. For example, abinding partner with a molecular weight of, for example, about 0.1 kDato about 20 kDa, can be tagged with, for example,succinimidyl-6-[a-methyl-a-(2-pyridyl-dithio)toluamido]hexanoate (SMPT),or dimethylpimelimidate (DMP), histidine, or a biotin molecule, as shownin FIG. 7B. Where the tag is biotin, the biotin molecule will bindsstrongly with streptavidin, which has a molecular weight of 60 kDa.Because the biotin/streptavidin interaction is highly specific, thestreptavidin amplifies the signal that would be produced only by thesmall binding partner by a factor of 60.

Detection sensitivity can be further enhanced through the use ofchemically derivatized small particles. “Nanoparticles” made ofcolloidal gold, various plastics, or glass with diameters of about 3-300nm can be coated with molecular species that will enable them tocovalently bind selectively to a binding partner. For example, as shownin FIG. 7C, nanoparticles that are covalently coated with streptavidincan be used to enhance the visibility of biotin-tagged binding partnerson the biosensor surface. While a streptavidin molecule itself has amolecular weight of 60 kDa, the derivatized bead can have a molecularweight of any size, including, for example, 60 KDa. Binding of a largebead will result in a large change in the optical density upon thebiosensor surface, and an easily measurable signal. This method canresult in an approximately 1000× enhancement in sensitivity resolution.

Surface-Relief Volume Diffractive Biosensors

Another embodiment of the invention is a biosensor that comprises volumesurface-relief volume diffractive structures (a SRVD biosensor). SRVDbiosensors have a surface that reflect predominantly at a particularnarrow band of optical wavelengths when illuminated with a broad band ofoptical wavelengths. Where specific binding substances and/or bindingpartners are immobilized on a SRVD biosensor, the reflected wavelengthof light is shifted. One-dimensional surfaces, such as thin filminterference filters and Bragg reflectors, can select a narrow range ofreflected or transmitted wavelengths from a broadband excitation source,however, the deposition of additional material, such as specific bindingsubstances and/or binding partners onto their upper surface results onlyin a change in the resonance linewidth, rather than the resonancewavelength. In contrast, SRVD biosensors have the ability to alter thereflected wavelength with the addition of material, such as specificbinding substances and/or binding partners to the surface.

A SRVD biosensor comprises a sheet material having a first and secondsurface. The first surface of the sheet material defines relief volumediffraction structures. A sheet material can be comprised of, forexample, plastic, glass, semiconductor wafer, or metal film.

A relief volume diffractive structure can be, for example, atwo-dimensional grating, as described above, or a three-dimensionalsurface-relief volume diffractive grating. The depth and period ofrelief volume diffraction structures are less than the resonancewavelength of light reflected from a biosensor.

A three-dimensional surface-relief volume diffractive grating can be,for example, a three-dimensional phase-quantized terraced surface reliefpattern whose groove pattern resembles a stepped pyramid. When such agrating is illuminated by a beam of broadband radiation, light Will becoherently reflected from the equally spaced terraces at a wavelengthgiven by twice the step spacing times the index of refraction of thesurrounding medium. Light of a given wavelength is resonantly diffractedor reflected from the steps that are a half-wavelength apart, and with abandwidth that is inversely proportional to the number of steps. Thereflected or diffracted color can be controlled by the deposition of adielectric layer so that a new wavelength is selected, depending on theindex of refraction of the coating.

A stepped-phase structure can be produced first in photoresist bycoherently exposing a thin photoresist film to three laser beams, asdescribed previously. See e.g., Cowen, “The recording and large scalereplication of crossed holographic grating arrays using multiple beaminterferometry,” in International Conference on the Application, Theory,and Fabrication of Periodic Structures, Diffraction Gratings, and MoirePhenomena II, Lerner, ed., Proc. Soc. Photo-Opt. Instrum. Eng., 503,120-129, 1984; Cowen, “Holographic honeycomb microlens,” Opt. Eng. 24,796-802 (1985); Cowen & Slafer, “The recording and replication ofholographic micropatterns for the ordering of photographic emulsiongrains in film systems,” J. Imaging Sci. 31, 100-107, 1987. Thenonlinear etching characteristics of photoresist are used to develop theexposed film to create a three-dimensional relief pattern. Thephotoresist structure is then replicated using standard embossingprocedures. For example, a thin silver film is deposited over thephotoresist structure to form a conducting layer upon which a thick filmof nickel can be electroplated. The nickel “master” plate is then usedto emboss directly into a plastic film, such as vinyl, that has beensoftened by heating or solvent.

The theory describing the design and fabrication of three-dimensionalphase-quantized terraced surface relief pattern that resemble steppedpyramids is described: Cowen, “Aztec surface-relief volume diffractivestructure,” J. Opt. Soc. Am. A, 7:1529 (1990).

An example of a three-dimensional phase-quantized terraced surfacerelief pattern is a pattern that resembles a stepped pyramid. Eachinverted pyramid is approximately 1 micron in diameter, preferably, eachinverted pyramid can be about 0.5 to about 5 microns diameter, includingfor example, about 1 micron. The pyramid structures can be close-packedso that a typical microarray spot with a diameter of 150-200 microns canincorporate several hundred stepped pyramid structures. The reliefvolume diffraction structures have a period of about 0.1 to about 1micron and a depth of about 0.1 to about 1 micron. FIG. 8 demonstrateshow individual microarray locations (with an entire microarray spotincorporating hundreds of pyramids now-represented by a single pyramidfor one microarray spot) can be optically queried to determine ifspecific binding substances or binding partners are adsorbed onto thesurface. When the structure is illuminated with white light, structureswithout significant bound material will reflect wavelengths determinedby the step height of the structure. When higher refractive indexmaterial, such as binding partners or specific binding substances, areincorporated over the reflective metal surface, the reflected wavelengthis modified to shift toward longer wavelengths. The color that isreflected from the terraced step structure is theoretically given astwice the step height times the index of refraction of a reflectivematerial that is coated onto the first surface of a sheet material of aSRVD biosensor. A reflective material can be, for example silver,aluminum, or gold.

One or more specific binding substances, as described above, areimmobilized on the reflective material of a SRVD biosensor. One or morespecific binding substances can be arranged in microarray of distinctlocations, as described above, on the reflective material. FIG. 9provides an example of a 9-element microarray biosensor. Many individualgrating structures, represented by small circles, lie within eachmicroarray spot. The microarray spots, represented by the largercircles, will reflect white light in air at a wavelength that isdetermined by the refractive index of material on their surface.Microarray locations with additional adsorbed material will havereflected wavelengths that are shifted toward longer wavelengths,represented by the larger circles.

Because the reflected wavelength of light from a SRVD biosensor isconfined to a narrow bandwidth, very small changes in the opticalcharacteristics of the surface manifest themselves in easily observedchanges in reflected wavelength spectra. The narrow reflection bandwidthprovides a surface adsorption sensitivity advantage compared toreflectance spectrometry on a flat surface.

A SRVD biosensor reflects light predominantly at a first single opticalwavelength when illuminated with a broad band of optical wavelengths,and reflects light at a second single optical wavelength when one ormore specific binding substances are immobilized on the reflectivesurface. The reflection at the second optical wavelength results fromoptical interference. A SRVD biosensor also reflects light at a thirdsingle optical wavelength when the one or more specific bindingsubstances are bound to their respective binding partners, due tooptical interference.

Readout of the reflected color can be performed serially by focusing amicroscope objective onto individual microarray spots and reading thereflected spectrum, or in parallel by, for example, projecting thereflected image of the microarray onto a high resolution color CCDcamera.

A SRVD biosensor can be manufactured by, for example, producing a metalmaster plate, and stamping a relief volume diffractive structure into,for example, a plastic material like vinyl. After stamping, the surfaceis made reflective by blanket deposition of, for example, a thin metalfilm such as gold, silver, or aluminum. Compared to MEMS-basedbiosensors that rely upon photolithography, etching, and wafer bondingprocedures, the manufacture of a SRVD biosensor is very inexpensive.

Liquid-Containing Vessels

A SWS or SRVD biosensor of the invention can comprise an inner surface,for example, a bottom surface of a liquid-containing vessel. Aliquid-containing vessel can be, for example, a microtiter plate well, atest tube, a petri dish, or a microfluidic channel. One embodiment ofthis invention is a SWS or SRVD biosensor that is incorporated into anytype of microtiter plate. For example, a SWS biosensor or SRVD biosensorcan be incorporated into the bottom surface of a microtiter plate byassembling the walls of the reaction vessels over the resonantreflection surface, as shown in FIG. 10, so that each reaction “spot”can be exposed to a distinct test sample. Therefore, each individualmicrotiter plate well can act as a separate reaction vessel. Separatechemical reactions can, therefore, occur within adjacent wells withoutintermixing reaction fluids and chemically distinct test solutions canbe applied to individual wells.

Several methods for attaching a biosensor of the invention to the bottomsurface of bottomless microtiter plates can be used, including, forexample, adhesive attachment, ultrasonic welding, and laser welding.

The most common assay formats for pharmaceutical high-throughputscreening laboratories, molecular biology research laboratories, anddiagnostic assay laboratories are microtiter plates. The plates arestandard-sized plastic cartridges that can contain 96, 384, or 1536individual reaction vessels arranged in a grid. Due to the standardmechanical configuration of these plates, liquid dispensing, roboticplate handling, and detection systems are designed to work with thiscommon format. A biosensor of the invention can be incorporated into thebottom surface of a standard microtiter plate. See, e g., FIG. 10.Because the biosensor surface can be fabricated in large areas, andbecause the readout system does not make physical contact with thebiosensor surface, an arbitrary number of individual biosensor areas canbe defined that are only limited by the focus resolution of theillumination optics and the x-y stage that scans theillumination/detection probe across the biosensor surface.

Holding Fixtures

Any number of biosensors that are, for example, about 1 mm² to about 5mm², and preferably less than about 3×3 mm² can be arranged onto aholding fixture that can simultaneously dip the biosensors into separateliquid-containing vessels, such as wells of a microtiter plate, forexample, a 96- , 384-, or 1536-well microtiter plate. See e.g., FIG. 11.Each of the biosensors can contain multiple distinct locations. Aholding fixture has one or more biosensors attached to the holdingfixture so that each individual biosensor can be lowered into a separateliquid-containing vessel. A holding fixture can comprise plastic, epoxyor metal. For example, 50, 96, 384, or 1,000, or 1,536 biosensors can bearranged on a holding fixture, where each biosensor has 25, 100, 500, or1,000 distinct locations. As an example, where 96 biosenors are attachedto a holding fixture and each biosensor comprises 100 distinctlocations, 9600 biochemical assays can be performed simultaneously.

Methods of Using SWS and SRVD Biosensors

SWS and SRVD biosensors of the invention can be used to study one or anumber of specific binding substance/binding partner interactions inparallel. Binding of one or more specific binding substances to theirrespective binding partners can be detected, without the use of labels,by applying one or more binding partners to a SWS or SRVD biosensor thathave one or more specific binding substances immobilized on theirsurfaces. A SWS biosensor is illuminated with light and a maxima inreflected wavelength, or a minima in transmitted wavelength of light isdetected from the biosensor. If one or more specific binding substanceshave bound to their respective binding partners, then the reflectedwavelength of light is shifted as compared to a situation where one ormore specific binding substances have not bound to their respectivebinding partners. Where a SWS biosensor is coated with an array ofdistinct locations containing the one or more specific bindingsubstances, then a maxima in reflected wavelength or minima intransmitted wavelength of light is detected from each distinct locationof the biosensor.

A SRVD biosensor is illuminated with light after binding partners havebeen added and the reflected wavelength of light is detected from thebiosensor. Where one or more specific binding substances have bound totheir respective binding partners, the reflected wavelength of light isshifted.

In one embodiment of the invention, a variety of specific bindingsubstances, for example, antibodies, can be immobilized in an arrayformat onto a biosensor of the invention. The biosensor is thencontacted with a test sample of interest comprising binding partners,such as proteins. Only the proteins that specifically bind to theantibodies immobilized on the biosensor remain bound to the biosensor.Such an approach is essentially a large-scale version of anenzyme-linked immunosorbent assay; however, the use of an enzyme orfluorescent label is not required.

The Activity of an enzyme can be detected by applying one or moreenzymes to a SWS or SRVD biosensor to which one or more specific bindingsubstances have been immobilized. The biosensor is washed andilluminated with light. The reflected wavelength of light is detectedfrom the biosensor. Where the one or more enzymes have altered the oneor more specific binding substances of the biosensor by enzymaticactivity, the reflected wavelength of light is shifted.

Additionally, a test sample, for example, cell lysates containingbinding partners can be applied to a biosensor of the invention,followed by washing to remove unbound material. The binding partnersthat bind to a biosensor can be eluted from the biosensor and identifiedby, for example, mass spectrometry. Optionally, a phage DNA displaylibrary can be applied to a biosensor of the invention followed bywashing to remove unbound material. Individual phage particles bound tothe biosensor can be isolated and the inserts in these phage particlescan then be sequenced to determine the identity of the binding partner.

For the above applications, and in particular proteomics applications,the ability to selectively bind material, such as binding partners froma test sample onto a biosensor of the invention, followed by the abilityto selectively remove bound material from a distinct location of thebiosensor for further analysis is advantageous. Biosensors of theinvention are also capable of detecting and quantifying the amount of abinding partner from a sample that is bound to a biosensor arraydistinct location by measuring the shift in reflected wavelength oflight. For example, the wavelength shift at one distinct biosensorlocation can be compared to positive and negative controls at otherdistinct biosensor locations to determine the amount of a bindingpartner that is bound to a biosensor array distinct location.

SWS and Electrically Conducting Material

An optional biosensor structure can further enable a biosensor array toselectively attract or repel binding partners from individual distinctlocations on a biosensor. As is well known in the art, an electromotiveforce can be applied to biological molecules such as nucleic acids andamino acids subjecting them to an electric field. Because thesemolecules are electronegative, they are attracted to a positivelycharged electrode and repelled by a negatively charged electrode.

A grating structure of a resonant optical biosensor can be built usingan electrically conducting material rather than an electricallyinsulating material. An electric field can be applied near the biosensorsurface. Where a grating operates as both a resonant reflector biosensorand as an electrode, the grating comprises a material that is bothoptically transparent near the resonant wavelength, and has lowresistivity. In one embodiment of the invention, the material is indiumtin oxide, InSn_(x)O_(1-x) (ITO). ITO is commonly used to producetransparent electrodes for flat panel optical displays, and is thereforereadily available at low cost on large glass sheets. The refractiveindex of ITO can be adjusted by controlling x, the fraction of Sn thatis present in the material. Because the liquid test sample solution willhave mobile ions (and will therefore be an electrical conductor) it isnecessary for the ITO electrodes to be coated with an insulatingmaterial. For the resonant optical biosensor, a grating layer is coatedwith a layer with lower refractive index material. Materials such ascured photoresist (n=1.65), cured optical epoxy (n=1.5), and glass(n=1.4-1.5) are strong electrical insulators that also have a refractiveindex that is lower than ITO (n=2.0-2.65). A cross-sectional diagram ofa biosensor that incorporates an ITO grating is shown in FIG. 48. n₁represents the refractive index of an electrical insulator. n₂represents the refractive index of a two-dimensional grating. t₁represents the thickness of the electrical insulator. t₂ represents thethickness of the two-dimensional grating. n_(bio) represents therefractive index of one or more specific binding substances and t_(BIO)represents the thickness of the one or more specific binding substances.

A grating can be a continuous sheet of ITO that contains an array ofregularly spaced holes. The holes are filled in with an electricallyinsulating material, such as cured photoresist. The electricallyinsulating layer overcoats the ITO grating so that the upper surface ofthe structure is completely covered with electrical insulator, and sothat the upper surface is substantially flat. When the biosensor isilluminated with light a resonant grating effect is produced on thereflected radiation spectrum. The depth and the period of the gratingare less than the wavelength of the resonant grating effect.

As shown in FIG. 12 and FIG. 13, a single electrode can comprise aregion that contains many grating periods. Building two or more separategrating regions on the same substrate surface creates an array ofbiosensor electrodes. Electrical contact to each biosensor electrode isprovided using an electrically conducting trace that is built from thesame material as the conductor within the biosensor electrode. Theconducting trace is connected to a voltage source that can apply anelectrical potential to the electrode. To apply an electrical potentialto the biosensor that is capable of attracting or repelling a moleculenear the electrode surface, a biosensor upper surface can be immersed ina liquid sample as shown in FIG. 14. A “common” electrode can be placedwithin the sample liquid, and a voltage can be applied between oneselected biosensor electrode region and the common electrode. In thisway, one, several, or all electrodes can be activated or inactivated ata given time. FIG. 15 illustrates the attraction of electronegativemolecules to the biosensor surface when a positive voltage is applied tothe electrode, while FIG. 16 illustrates the application of a repellingforce such as a reversed electrical charge to electronegative moleculesusing a negative electrode voltage.

Detection Systems

A detection system can comprise a biosensor of the invention, a lightsource that directs light to the biosensor, and a detector that detectslight reflected from the biosensor. In one embodiment, it is possible tosimplify the readout instrumentation by the application of a filter sothat only positive results over a determined threshold trigger adetection.

A light source can illuminate a biosensor from its top surface, i.e.,the surface to which one or more specific binding substances areimmobilized or from its bottom surface. By measuring the shift inresonant wavelength at each distinct location of a biosensor of theinvention, it is possible to determine which distinct locations havebinding partners bound to them. The extent of the shift can be used todetermine the amount of binding partners in a test sample and thechemical affinity between one or more specific binding substances andthe binding partners of the test sample.

A biosensor of the invention can be illuminated twice. The firstmeasurement determines the reflectance spectra of one or more distinctlocations of a biosensor array with one or more specific bindingsubstances immobilized on the biosensor. The second measurementdetermines the reflectance spectra after one or more binding partnersare applied to a biosensor. The difference in peak wavelength betweenthese two measurements is a measurement of the amount of bindingpartners that have specifically bound to a biosensor or one or moredistinct locations of a biosensor. This method of illumination cancontrol for small nonuniformities in a surface of a biosensor that canresult in regions with slight variations in the peak resonantwavelength. This method can also control for varying concentrations ormolecular weights of specific binding substances immobilized on abiosensor

Computer simulation can be used to determine the expected dependencebetween a peak resonance wavelength and an angle of incidentillumination. A biosensor structure as shown in FIG. 1 can be forpurposes of demonstration. The substrate chosen was glass(n_(substrate)=1.50). The grating is a two-dimensional pattern ofsilicon nitride squares (t₂=180 nm, n₂=2.01 (n=refractive index),k₂=0.001 (k=absorption coefficient)) with a period of 510 nm, and afilling factor of 56.2% (i.e., 56.2% of the surface is covered withsilicon nitride squares while the rest is the area between the squares).The areas between silicon nitride squares are filled with a lowerrefractive index material. The same material also covers the squares andprovides a uniformly flat upper surface. For this simulation, a glasslayer was selected (n₁=1.40) that covers the silicon nitride squares byt₂=100 nm.

The reflected intensity as a function of wavelength was modeled usingGSOLVER software, which utilizes full 3-dimensional vector code usinghybrid Rigorous Coupled Wave Analysis and Modal analysis. GSOLVERcalculates diffracted fields and diffraction efficiencies from planewave illumination of arbitrarily complex grating structures. Theillumination can be from any incidence and any polarization.

FIG. 19 plots the dependence of the peak resonant wavelength upon theincident illumination angle. The simulation shows that there is a strongcorrelation between the angle of incident light, and the peak wavelengththat is measured. This result implies that the collimation of theilluminating beam, and the alignment between the illuminating beam andthe reflected beam will directly affect the resonant peak linewidth thatis measured. If the collimation of the illuminating beam is poor, arange illuminating angles will be incident on the biosensor surface, anda wider resonant peak will be measured than if purely collimated lightwere incident.

Because the lower sensitivity limit of a biosensor is related to theability to determine the peak maxima, it is important to measure anarrow resonant peak. Therefore, the use of a collimating illuminationsystem with the biosensor provides for the highest possible sensitivity.

One type of detection system for illuminating the biosensor surface andfor collecting the reflected light is a probe containing, for example,six illuminating optical fibers that are connected to a light source,and a single collecting optical fiber connected to a spectrometer. Thenumber of fibers is not critical, any number of illuminating orcollecting fibers are possible. The fibers are arranged in a bundle sothat the collecting fiber is in the center of the bundle, and issurrounded by the six illuminating fibers. The tip of the fiber bundleis connected to a collimating lens that focuses the illumination ontothe surface of the biosensor.

In this probe arrangement, the illuminating and collecting fibers areside-by-side. Therefore, when the collimating lens is correctly adjustedto focus light onto the biosensor surface, one observes six clearlydefined circular regions of illumination, and a central dark region.Because the biosensor does not scatter light, but rather reflects acollimated beam, no light is incident upon the collecting fiber, and noresonant signal is observed. Only by defocusing the collimating lensuntil the six illumination regions overlap into the central region isany light reflected into the collecting fiber. Because only defocused,slightly uncollimated light can produce a signal, the biosensor is notilluminated with a single angle of incidence, but with a range ofincident angles. The range of incident angles results in a mixture ofresonant wavelengths due to the dependence shown in FIG. 19. Thus, widerresonant peaks are measured than might otherwise be possible.

Therefore, it is desirable for the illuminating and collecting fiberprobes to spatially share the same optical path. Several methods can beused to co-locate the illuminating and collecting optical paths. Forexample, a single illuminating fiber, which is connected at its firstend to a light source that directs light at the biosensor, and a singlecollecting fiber, which is connected at its first end to a detector thatdetects light reflected from the biosensor, can each be connected attheir second ends to a third fiber probe that can act as both anilluminator and a collector. The third fiber probe is oriented at anormal angle of incidence to the biosensor and supportscounter-propagating illuminating and reflecting optical signals. Anexample of such a detection system is shown in FIG. 18.

Another method of detection involves the use of a beam splitter thatenables a single illuminating fiber, which is connected to a lightsource, to be oriented at a 90 degree angle to a collecting fiber, whichis connected to a detector. Light is directed through the illuminatingfiber probe into the beam splitter, which directs light at thebiosensor. The reflected light is directed back into the beam splitter,which directs light into the collecting fiber probe. An example of sucha detection device is shown in FIG. 20. A beam splitter allows theilluminating light and the reflected light to share a common opticalpath between the beam splitter and the biosensor, so perfectlycollimated light can be used without defocusing.

Angular Scanning

Detection systems of the invention are based on collimated white lightillumination of a biosensor surface and optical spectroscopy measurementof the resonance peak of the reflected beam. Molecular binding on thesurface of a biosensor is indicated by a shift in the peak wavelengthvalue, while an increase in the wavelength corresponds to an increase inmolecular absorption.

As shown in theoretical modeling and experimental data, the resonancepeak wavelength is strongly dependent on the incident angle of thedetection light beam. FIG. 19 depicts this dependence as modeled for abiosensor of the invention. Because of the angular dependence of theresonance peak wavelength, the incident white light needs to be wellcollimated. Angular dispersion of the light beam broadens the resonancepeak, and reduces biosensor detection sensitivity. In addition, thesignal quality from the spectroscopic measurement depends on the powerof the light source and the sensitivity of the detector. In order toobtain a high signal-to-noise ratio, an excessively long integrationtime for each detection location can be required, thus lengtheningoverall time to readout a biosensor plate. A tunable laser source can beused for detection of grating resonance, but is expensive.

In one embodiment of the invention, these disadvantages are addressed byusing a laser beam for illumination of a biosensor, and a light detectorfor measurement of reflected beam power. A scanning mirror device can beused for varying the incident angle of the laser beam, and an opticalsystem is used for maintaining collimation of the incident laser beam.See, e.g., “Optical Scanning” (Gerald F. Marchall ed., Marcel Dekker(1991). Any type of laser scanning can be used. For example, a scanningdevice that can generate scan lines at a rate of about 2 lines to about1,000 lines per second is useful in the invention. In one embodiment ofthe invention, a scanning device scans from about 50 lines to about 300lines per second.

In one embodiment, the reflected light beam passes through part of thelaser scanning optical system, and is measured by a single lightdetector. The laser source can be a diode laser with a wavelength of,for example, 780 nm, 785 nm, 810 nm, or 830 nm. Laser diodes such asthese are readily available at power levels up to 150 mW, and theirwavelengths correspond to high sensitivity of Si photodiodes. Thedetector thus can be based on photodiode biosensors. An example of sucha detection system is shown in FIG. 52. A light source (100) provideslight to a scanner device (200), which directs the light into an opticalsystem (300) The optical system (300) directs light to a biosensor (400)Light is reflected from the biosensor (400) to the optical system (300),which then directs the light into a light signal detector (500). Oneembodiment of a detection system is shown in FIG. 21, which demonstratesthat while the scanning mirror changes its angular position, theincident angle of the laser beam on the surface changes by nominallytwice the mirror angular displacement. The scanning mirror device can bea linear galvanometer, operating at a frequency of about 2 Hz up toabout 120 Hz, and mechanical scan angle of about 10 degrees to about 20degrees. In this example, a single scan can be completed within about 10msec. A resonant galvanometer or a polygon scanner can also be used. Theexample shown in FIG. 21 includes a simple optical system for angularscanning. It consists of a pair of lenses with a common focal pointbetween them. The optical system can be designed to achieve optimizedperformance for laser collimation and collection of reflected lightbeam.

The angular resolution depends on the galvanometer specification, andreflected light sampling frequency. Assuming galvanometer resolution of30 arcsec mechanical, corresponding resolution for biosensor angularscan is 60 arcsec, i.e. 0.017 degree. In addition, assume a samplingrate of 100 ksamples/sec, and 20 degrees scan within 10 msec. As aresult, the quantization step is 20 degrees for 1000 samples, i.e. 0.02degree per sample. In this example, a resonance peak width of 0.2degree, as shown by Peng and Morris (Experimental demonstration ofresonant anomalies in diffraction from two-dimensional gratings, OpticsLett., 21:549 (1996)), will be covered by 10 data points, each of whichcorresponds to resolution of the detection system.

The advantages of such a detection system includes: excellentcollimation of incident light by a laser beam, high signal-to-noiseratio due to high beam power of a laser diode, low cost due to a singleelement light detector instead of a spectrometer, and high resolution ofresonance peak due to angular scanning.

Fiber Probe Biosensor

A biosensor of the invention can occur on the tip of a multi-mode fiberoptic probe. This fiber optic probe allows for in vivo detection ofbiomarkers for diseases and conditions, such as, for example, cardiacartery disease, cancer, inflammation, and sepsis. A single biosensorelement (comprising, for example, several hundred grating periods) canbe fabricated into the tip of a fiber optic probe, or fabricated from aglass substrate and attached to the tip of a fiber optic probe. See FIG.17. A single fiber is used to provide illumination and measure resonantreflected signal.

For example, a fiber probe structure similar to that shown in FIG. 18can be used to couple an illuminating fiber and detecting fiber into asingle counterpropagating fiber with a biosensor embedded or attached toits tip. The fiber optic probe is inserted into a mammalian body, forexample, a human body. Illumination and detection of a reflected signalcan occur while the probe is inserted in the body.

Mathematical Resonant Peak Determination

The sensitivity of a biosensor is determined by the shift in thelocation of the resonant peak when material is bound to the biosensorsurface. Because of noise inherent in the spectrum, it is preferable touse a procedure for determining an analytical curve—the turning point(i.e., peak) of which is well defined. Furthermore, the peakcorresponding to an analytic expression can be preferably determined togreater than sub-sampling-interval accuracy, providing even greatersensitivity.

One embodiment of the invention provides a method for determining alocation of a resonant peak for a binding partner in a resonantreflectance spectrum with a colormetric resonant biosensor. The methodcomprises selecting a set of resonant reflectance data for a pluralityof colormetric resonant biosensors or a plurality of biosensor distinctlocations. The set of resonant reflectance data is collected byilluminating a colormetric resonant diffractive grating surface with alight source and measuring reflected light at a pre-determinedincidence. The colormetric resonant diffractive grating surface is usedas a surface binding platform for one or more specific bindingsubstances such that binding partners can be detected without use of amolecular label.

The step of selecting a set of resonant reflectance data can includeselecting a set of resonant reflectance data:x_(i) and y_(i) for i=1, 2, 3, . . . n,wherein x_(i) is where a first measurement includes a first reflectancespectra of one or more specific binding substances attached to thecolormetric resonant diffractive grating surface, y_(i) and a secondmeasurement and includes a second reflectance spectra of the one or morespecific binding substances after a plurality of binding partners areapplied to colormetric resonant diffractive grating surface includingthe one or more specific binding substances, and n is a total number ofmeasurements collected.

The set of resonant reflectance data includes a plurality of sets of twomeasurements, where a first measurement includes a first reflectancespectra of one or more specific binding substances that are attached tothe colormetric resonant diffractive grating surface and a secondmeasurement includes a second reflectance spectra of the one or morespecific binding substances after one or more binding partners areapplied to the colormetric resonant diffractive grating surfaceincluding the one or more specific binding substances. A difference in apeak wavelength between the first and second measurement is ameasurement of an amount of binding partners that bound to the one ormore specific binding substances. A sensitivity of a colormetricresonant biosensor can be determined by a shift in a location of aresonant peak in the plurality of sets of two measurements in the set ofresonant reflectance data.

A maximum value for a second measurement from the plurality of sets oftwo measurements is determined from the set of resonant reflectance datafor the plurality of binding partners, wherein the maximum valueincludes inherent noise included in the resonant reflectance data. Amaximum value for a second measurement can include determining a maximumvalue y_(k) such that:(y _(k) >=y _(i)) for all i≠k.

It is determined whether the maximum value is greater than apre-determined threshold. This can be calculated by, for example,computing a mean of the set of resonant reflectance data; computing astandard deviation of the set of resonant reflectance data; anddetermining whether ((y_(k)−mean)/standard deviation) is greater than apre-determined threshold. The pre-determined threshold is determined bythe user. The user will determine what amount of sensitivity is desiredand will set the pre-determined threshold accordingly.

If the maximum value is greater than a pre-determined threshold acurve-fit region around the determined maximum value is defined. Thestep of defining a curve-fit region around the determined maximum valuecan include, for example:

defining a curve-fit region of (2w+1) bins, wherein w is apre-determined accuracy value;

extracting (x_(i,), k−w<=i<=k+w); and

extracting (y_(i,), k−w<=i<=k+w).

A curve-fitting procedure is performed to fit a curve around thecurve-fit region, wherein the curve-fitting procedure removes apre-determined amount of inherent noise included in the resonantreflectance data. A curve-fitting procedure can include, for example:

computing g_(i)=ln y_(i);

performing a 2^(nd) order polynomial fit on g_(i) to obtain g′_(i)defined on(x _(i,) , k−w<=i<=k+w);

determining from the 2^(nd) order polynomial fit coefficients a, b and cof for (ax²+bx+c)−; and

computing y′_(i)=e^(g′i).

The location of a maximum resonant peak is determined on the fittedcurve, which can include, for example, determining a location of maximumreasonant peak (x_(p)=(−b)/2a). A value of the maximum resonant peak isdetermined, wherein the value of the maximum resonant peak is used toidentify an amount of biomolecular binding of the one or more specificbinding substances to the one or more binding partners. A value of themaximum resonant peak can include, for example, determining the valuewith of x_(p) at y′_(p).

One embodiment of the invention comprises a computer readable mediumhaving stored therein instructions for causing a processor to execute amethod for determining a location of a resonant peak for a bindingpartner in a resonant reflectance spectrum with a colormetric resonantbiosensor. A computer readable medium can include, for example, magneticdisks, optical disks, organic memory, and any other volatile (e.g.,Random Access Memory (“RAM”)) or non-volatile (e.g., Read-Only Memory(“ROM”)) mass storage system readable by the processor. The computerreadable medium includes cooperating or interconnected computer readablemedium, which exist exclusively on a processing system or to bedistributed among multiple interconnected processing systems that can belocal or remote to the processing system.

The following are provided for exemplification purpose only and are notintended to limit the scope of the invention described in broad termsabove. All references cited in this disclosure are incorporated hereinby reference.

EXAMPLE 1

Fabrication of a SWS Biosensor

An example of biosensor fabrication begins with a flat glass substratethat is coated with a thin layer (180 nm) of silicon nitride byplasma-enhanced chemical vapor deposition (PECVD).

The desired structure is first produced in photoresist by coherentlyexposing a thin photoresist film to three laser beams, as described inpreviously (Cowen, “The recording and large scale replication of crossedholographic grating arrays using multiple beam interferometry,” inInternational Conference on the Application, Theory, and Fabrication ofPeriodic Structures, Diffraction Gratings, and Moire Phenomena II, J. M.Lerner, ed, Proc. Soc. Photo-Opt. Instrum. Eng., 503, 120-129, 1984;Cowen, “Holographic honeycomb microlens,” Opt. Eng. 24, 796-802 (1985);Cowen & Slafer, “The recording and replication of holographicmicropatterns for the ordering of photographic emulsion grains in filmsystems,” J. Imaging Sci. 31, 100-107, 1987. The nonlinear etchingcharacteristics of photoresist are used to develop the exposed film tocreate a pattern of holes within a hexagonal grid, as shown in FIG. 22.The photoresist pattern is transferred into the silicon nitride layerusing reactive ion etching (RIE). The photoresist is removed, and acover layer of spin-on-glass (SOG) is applied (Honeywell ElectronicMaterials, Sunnyvale, Calif.) to fill in the open regions of the siliconnitride grating. The structure of the top surface of the finishedbiosensor is shown in FIG. 23. A photograph of finished parts are shownin FIG. 24.

EXAMPLE 2

A SRVD biosensor was prepared by making five circular diffuse gratingholograms by stamping a metal master plate into vinyl. The circularholograms were cut out and glued to glass slides. The slides were coatedwith 1000 angstroms of aluminum. In air, the resonant wavelength of thegrating is ˜380 nm, and therefore, no reflected color is visible. Whenthe grating is covered with water, a light blue reflection is observed.Reflected wavelength shifts are observable and measurable while thegrating is covered with a liquid, or if a specific binding substancesand/or binding partners cover the structure.

Both proteins and bacteria were immobilized onto the surface of a SRVDbiosensor at high concentration and the wavelength shift was measured.For each material, a 20 μl droplet is placed onto a biosensor distinctlocation and allowed to dry in air. At 1 μg/ml protein concentration, a20 μl droplet spreads out to cover a 1 cm diameter circle and depositsabout 2×10⁻⁸ grams of material. The surface density is 25.6 ng/mm².

For high concentration protein immobilization (biosensor 4) a 10 μldroplet of 0.8 g bovine serum albumin (BSA) in 40 ml DI H₂O is spreadout to cover a 1 cm diameter circle on the surface of a biosensor. Thedroplet deposits 0.0002 g of BSA, for a density of 2.5 e-6 g/mm². Afterprotein deposition, biosensor 4 has a green resonance in air.

For bacteria immobilization (biosensor 2) a 20 μl droplet of NECKborrelia Lyme Disease bacteria (1.8 e8 cfu/ml) was deposited on thesurface of a biosensor. After bacteria deposition, the biosensor looksgrey in air.

For low concentration protein immobilization (biosensor 6) a 10 μldroplet of 0.02% of BSA in DI H₂O (0.8 g BSA in 40 ml DI H₂O) is spreadout to cover a 1 cm diameter circle. The droplet deposits 0.000002 g ofBSA for a density of 2.5 e-8 g/mm². After protein deposition, biosensor6 looks grey in air.

In order to obtain quantitative data on the extent of surfacemodification resulting from the above treatments, the biosensors weremeasured using a spectrometer.

Because a green resonance signal was immediately visually observed onthe biosensor upon which high concentration BSA was deposited (biosensor4), it was measured in air. FIG. 25 shows two peaks at 540 nm and 550 nmin green wavelengths where none were present before protein deposition,indicating that the presence of a protein thin film is sufficient toresult in a strong shift in resonant wavelength of a surface reliefstructure.

Because no visible resonant wavelength was observed in air for the slideupon which a low concentration of protein was applied (biosensor 6), itwas measured with distilled water on surface and compared against abiosensor which had no protein treatment. FIG. 26 shows that theresonant wavelength for the slide with protein applied shifted to greencompared to a water-coated slide that had not been treated.

Finally, a water droplet containing Lyme Disease bacteria Borreliaburgdorferi was applied to a grating structure and allowed to dry in air(biosensor 2). Because no visually observed resonance occurred in airafter bacteria deposition, the biosensor was measured with distilledwater on the surface and compared to a water-coated biosensor that hadundergone no other treatment. As shown in FIG. 27, the application ofbacteria results in a resonant frequency shift to longer wavelengths.

EXAMPLE 3

Computer Model of Biosensor

To demonstrate the concept that a resonant grating structure can be usedas a biosensor by measuring the reflected wavelength shift that isinduced when biological material is adsorbed onto its surface, thestructure shown in FIG. 1 was modeled by computer. For purposes ofdemonstration, the substrate chosen was glass (n_(substrate)=1.50). Thegrating is a two-dimensional pattern of silicon nitride squares (t₂=180mn, n₂=2.01, k₂=0.001) with a period of 510 nm, and a filling factor of56.2% (i.e. 56.2% of the surface is covered with silicon nitride squareswhile the rest is the area between the squares). The areas betweensilicon nitride squares are filled with a lower refractive indexmaterial. The same material also covers the squares and provides auniformly flat upper surface. For this simulation, a glass layer wasselected (n₁=1.40) that covers the silicon nitride squares by t₂=100 nm.To observe the effect on the reflected wavelength of this structure withthe deposition of biological material, variable thicknesses of protein(n_(bio)=1.5) were added above the glass coating layer.

The reflected intensity as a function of wavelength was modeled usingGSOLVER software, which utilizes full 3-dimensional vector code usinghybrid Rigorous Coupled Wave Analysis and Modal analysis. GSOLVERcalculates diffracted fields and diffraction efficiencies from planewave illumination of arbitrarily complex grating structures. Theillumination may be from any incidence and any polarization.

The results of the computer simulation are shown in FIG. 28 and FIG. 29.As shown in FIG. 28, the resonant structure allows only a singlewavelength, near 780 nm, to be reflected from the surface when noprotein is present on the surface. Because the peak width athalf-maximum is ˜1.5 nm, resonant wavelength shifts of ˜0.2 nm will beeasily resolved. FIG. 28 also shows that the resonant wavelength shiftsto longer wavelengths as more protein is deposited on the surface of thestructure. Protein thickness changes of 2 nm are easily observed. FIG.29 plots the dependence of resonant wavelength on the protein coatingthickness. A near linear relationship between protein thickness andresonant wavelength is observed, indicating that this method ofmeasuring protein adsorption can provide quantitative data. For thesimulated structure, FIG. 29 shows that the wavelength shift responsebecomes saturated when the total deposited protein layer exceeds ˜250nm. This upper limit for detection of deposited material providesadequate dynamic range for any type of biomolecular assay.

EXAMPLE 4

Computer Model of Biosensor

In another embodiment of the invention a biosensor structure shown inFIG. 30 was modeled by computer. For purposes of demonstration, thesubstrate chosen was glass n_(substrate)=1.454 coated with a layer ofhigh refractive index material such as silicon nitride, zinc sulfide,tantalum oxide, or titanium dioxide. In this case, silicon nitride(t₃=90 nm, n₃=2.02) was used. The grating is two-dimensional pattern ofphotoresist squares (t₂=90 nm, n₂=1.625) with a period of 510 nm, and afilling factor of 56.2% (i.e. 56.2% of the surface is covered withphotoresist squares while the rest is the area between the squares). Theareas between photoresist squares are filled with a lower refractiveindex material such as glass, plastic, or epoxy. The same material alsocovers the squares and provides a uniformly flat upper surface. For thissimulation, a glass layer was selected (n₁=1.45) that covers thephotoresist squares by t₂=100 nm. To observe the effect on the reflectedwavelength of this structure with the deposition of a specific bindingsubstance, variable thicknesses of protein (n_(bio)=1.5) were addedabove the glass coating layer.

The reflected intensity as a function of wavelength was modeled usingGSOLVER software, which utilizes full 3-dimensional vector code usinghybrid Rigorous Coupled Wave Analysis and Modal analysis. GSOLVERcalculates diffracted fields and diffraction efficiencies from planewave illumination of arbitrarily complex grating structures. Theillumination may be from any incidence and any polarization.

The results of the computer simulation are shown in FIG. 31 and FIG. 32.The resonant structure allows only a single wavelength, near 805 nm, tobe reflected from the surface when no protein is present on the surface.Because the peak width at half-maximum is <0.25 nm, resonant wavelengthshifts of 1.0 nm will be easily resolved. FIG. 31 also shows that theresonant wavelength shifts to longer wavelengths as more protein isdeposited on the surface of the structure. Protein thickness changes of1 nm are easily observed. FIG. 32 plots the dependence of resonantwavelength on the protein coating thickness. A near linear relationshipbetween protein thickness and resonant wavelength is observed,indicating that this method of measuring protein adsorption can providequantitative data.

EXAMPLE 5

Sensor Readout Instrumentation

In order to detect reflected resonance, a white light source canilluminate a ˜1 mm diameter region of a biosensor surface through a 400micrometer diameter fiber optic and a collimating lens, as shown in FIG.33. Smaller or larger areas may be sampled through the use ofillumination apertures and different lenses. A group of six detectionfibers are bundled around the illumination fiber for gathering reflectedlight for analysis with a spectrometer (Ocean Optics, Dunedin, Fla.).For example, a spectrometer can be centered at a wavelength of 800 nm,with a resolution of ˜0.14 nm between sampling bins. The spectrometerintegrates reflected signal for 25-75 msec for each measurement. Thebiosensor sits upon an x-y motion stage so that different regions of thebiosensor surface can be addressed in sequence.

Equivalent measurements can be made by either illuminating the topsurface of device, or by illuminating through the bottom surface of thetransparent substrate. Illumination through the back is preferred whenthe biosensor surface is immersed in liquid, and is most compatible withmeasurement of the biosensor when it is incorporated into the bottomsurface of, for example, a microwell plate.

EXAMPLE 6

Demonstration of Resonant Reflection

FIG. 34 shows the resonant reflectance spectra taken from a biosensor asshown in FIG. 1 using the instrumentation described in Example 5. Thewavelength of the resonance (λ_(peak)=772.5 nm) compares with theresonant wavelength predicted by the computer model (λ_(peak)=781 nm),and the measured reflectance efficiency (51%) is comparable to thepredicted efficiency (70%). The greatest discrepancy between themeasured and predicted characteristics is the linewidth of the resonantpeak. The measured full-width at half maximum (FWHM) of the resonance is6 nm, while the predicted FWHM is 1.5 nm. As will be shown, the dominantsource of the larger measured FWHM is collimation of the illuminationoptics, which can easily be corrected.

As a basic demonstration of the resonant structure's ability to detectdifferences in the refractive index of materials in contact with itssurface, a biosensor was exposed to a series of liquids withwell-characterized optical properties. The liquids used were water,methanol, isopropyl alcohol, acetone, and DMF. A biosensor was placedface-down in a small droplet of each liquid, and the resonant wavelengthwas measured with a fiber illumination/detection probe facing thebiosensor's back side. Table 1 shows the calculated and measured peakresonant wavelength as a biosensor surface is exposed to liquids withvariable refractive index demonstrating the correlation between measuredand theoretical detection sensitivity. As shown in Table 1, the measuredresonant peak positions and measured resonant wavelength shifts arenearly identical to the predicted values. This example demonstrates theunderlying sensitivity of the biosensor, and validates the computermodel that predicts the wavelength shift due to changes in the materialin contact with the surface.

TABLE 1 Calculated Measured Peak Peak Wavelength Shift Wavelength ShiftSolution n (nm) (nm) (nm) (nm) Water 1.333 791.6 0 786.08 0 Isopropyl1.3776 795.9 4.3 789.35 3.27 Acetone 1.3588 794 2.4 788.22 2.14 Methanol1.3288 791.2 −0.4 785.23 −0.85 DMF 1.4305 802 10.4 796.41 10.33

Similarly, a biosensor is able to measure the refractive indexdifference between various buffer solutions. As an example, FIG. 35shows the variation in peak wavelength with the concentration of bovineserum albumin (BSA) in water. Resonance was measured with the biosensorplaced face-down in a droplet of buffer, and rinsed with water betweeneach measurement.

EXAMPLE 7

Immobilized Protein Detection

While the detection experiments shown in Example 6 demonstrate abiosensor's ability to measure small differences in refractive index ofliquid solutions, the biosensor is intended to measure specific bindingsubstances and binding partners that are chemically bound to thebiosensor surface. In order to demonstrate a biosensor's ability toquantify biomolecules on its surface, droplets of BSA dissolved in PBSat various concentrations were applied to a biosensor as shown inFIG. 1. The 3 μl droplets were allowed to dry in air, leaving a smallquantity of BSA distributed over a ˜2 mm diameter area. The peakresonant wavelength of each biosensor location was measured before andafter droplet deposition, and the peak wavelength shift was recorded.See FIG. 37.

EXAMPLE 8

Immobilization of One or More Specific Binding Substances

The following protocol was used on a colorimetric resonant reflectivebiosensor to activate the surface with amine functional groups. Aminegroups can be used as a general-purpose surface for subsequent covalentbinding of several types of linker molecules.

A biosensor of the invention is cleaned by immersing it into piranhaetch (70/30% (v/v) concentrated sulfuric acid/30% hydrogen peroxide) for12 hours. The biosensor was washed thoroughly with water. The biosensorwas dipped in 3% 3-aminopropyltriethoxysilane solution in dry acetonefor 1 minute and then rinsed with dry acetone and air-dried. Thebiosensor was then washed with water.

A semi-quantitative method is used to verify the presence of aminogroups on the biosensor surface. One biosensor from each batch ofamino-functionalized biosensors is washed briefly with 5 mL of 50 mMsodium bicarbonate, pH 8.5. The biosensor is then dipped in 5 mL of 50mM sodium bicarbonate, pH 8.5 containing 0.1 mMsulfo-succinimidyl-4-O-(4,4′-dimethoxytrityl)-butyrate (s-SDTB, Pierce,Rockford, Ill.) and shaken vigorously for 30 minutes. The s-SDTBsolution is prepared by dissolving 3.0 mg of s-SDTB in 1 mL of DMF anddiluting to 50 mL with 50 mM sodium bicarbonate, pH 8.5. After a 30minute incubation, the biosensor is washed three times with 20 mL ofddH₂O and subsequently treated with 5 mL 30% perchloric acid. Thedevelopment of an orange-colored solution indicates that the biosensorhas been successfully derivatized with amines; no color change isobserved for untreated glass biosensors.

The absorbance at 495 nm of the solution after perchloric acid treatmentfollowing the above procedure can be used as an indicator of thequantity of amine groups on the surface. In one set of experiment, theabsorbance was 0.627, 0.647, and 0.728 for Sigma slides, Cel-Associateslides, and in-house biosensor slides, respectively. This indicates thatthe level of NH₂ activation of the biosensor surface is comparable inthe activation commercially available microarray glass slides.

After following the above protocol for activating the biosensor withamine, a linker molecule can be attached to the biosensor. Whenselecting a cross-linking reagent, issues such as selectivity of thereactive groups, spacer arm length, solubility, and cleavability shouldbe considered. The linker molecule, in turn, binds the specific bindingsubstance that is used for specific recognition of a binding partner. Asan example, the protocol below has been used to bind a biotin linkermolecule to the amine-activated biosensor.

Protocol for Activating Amine-Coated Biosensor with Biotin

Wash an amine-coated biosensor with PBS (pH 8.0) three times. Preparesulfo-succinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin,Pierce, Rockford, Ill.) solution in PBS buffer (pH 8) at 0.5 mg/mlconcentration. Add 2 ml of the sulfo-NHS-LC-biotin solution to eachamine-coated biosensor and incubate at room temperature for 30 min. Washthe biosensor three times with PBS (pH 8.0). The sulfo-NHS-LC-biotinlinker has a molecular weight of 556.58 and a length of 22.4 Å. Theresulting biosensors can be used for capturing avidin or strepavidinmolecules.

Protocol for Activating Amine-Coated Biosensor with Aldehyde

Prepare 2.5% glutaraldehyde solution in 0.1 M sodium phosphate, 0.05%sodium azide, 0.1% sodium cyanoborohydride, pH 7.0. Add 2 ml of thesulfo-NHS-LC-biotin solution to each amine-coated biosensor and incubateat room temperature for 30 min. Wash the biosensor three times with PBS(pH 7.0). The glutaraldehyde linker has a molecular weight of 100.11.The resulting biosensors can be used for binding proteins and otheramine-containing molecules. The reaction proceeds through the formationof Schiff bases, and subsequent reductive amination yields stablesecondary amine linkages. In one experiment, where a coated aldehydeslide made by the inventors was compared to a commercially availablealdehyde slide (Cel-Associate), ten times higher binding of streptavidinand anti-rabbit IgG on the slide made by the inventors was observed.

Protocol for Activating Amine-coated Biosensor with NHS

25 mM N,N′-disuccinimidyl carbonate (DSC, Sigma Chemical Company, St.Louis, Mo.) in sodium carbonate buffer (pH 8.5) was prepared. 2 ml ofthe DSC solution was added to each amine-coated biosensor and incubatedat room temperature for 2 hours. The biosensors were washed three timeswith PBS (pH 8.5). A DSC linker has a molecular weight of 256.17.Resulting biosensors are used for binding to hydroxyl- oramine-containing molecules. This linker is one of the smallesthomobifunctional NHS ester cross-linking reagents available.

In addition to the protocols defined above, many additional surfaceactivation and molecular linker techniques have been reported thatoptimize assay performance for different types of biomolecules. Mostcommon of these are amine surfaces, aldehyde surfaces, and nickelsurfaces. The activated surfaces, in turn, can be used to attach severaldifferent types of chemical linkers to the biosensor surface, as shownin Table 2. While the amine surface is used to attach several types oflinker molecules, the aldehyde surface is used to bind proteinsdirectly, without an additional linker. A nickel surface is usedexclusively to bind molecules that have an incorporated histidine(“his”) tag. Detection of “his-tagged” molecules with a Nickel activatedsurface is well known (Sigal et al., Anal. Chem. 68, 490 (1996)).

Table 2 demonstrates an example of the sequence of steps that are usedto prepare and use a biosensor, and various options that are availablefor surface activation chemistry, chemical linker molecules, specificbinding substances and binding partners molecules. Opportunities alsoexist for enhancing detected signal through amplification with largermolecules such as HRP or streptavidin and the use of polymer materialssuch as dextran or TSPS to increase surface area available for molecularbinding.

TABLE 2 Label Bare Surface Linker Receptor Detected Molecule SensorActivation Molecule Molecule Material (Optional) Glass Amino SMPT SmPeptide Enhance Polymers Aldehyde NHS-Biotin m'cules Med sensi- optionalto Ni DMP Peptide Protein tivity enhance NNDC Med Lrg 1000× sensitivityHis-tag Protein Protein• HRP 2-5× Others . . . Lrg IgG Strep- DextranProtein• Phage tavidin TSPS IgG Cell cDNA cDNA

EXAMPLE 9

IgG Assay

As an initial demonstration for detection of biochemical binding, anassay was performed in which a biosensor was prepared by activation withthe amino surface chemistry described in Example 8 followed byattachment of a biotin linker molecule. The biotin linker is used tocovalently bond a streptavidin receptor molecule to the surface byexposure to a 50 μg/ml concentration solution of streptavidin in PBS atroom temperature for 2-4 hours. The streptavidin receptor is capable ofbinding any biotinylated protein to the biosensor surface. For thisexample, 3 μl droplets of biotinylated anti-human IgG in phosphatebuffer solution (PBS) were deposited onto 4 separate locations on thebiosensor surface at a concentration of 200 μg/ml. The solution wasallowed to incubate on the biosensor for 60 min before rinsingthoroughly with PBS. The peak resonant wavelength of the 4 locationswere measured after biotin activation, after streptavidin receptorapplication, and after ah-IgG binding. FIG. 37 shows that the additionof streptavidin and ah-IgG both yield a clearly measurable increase inthe resonant wavelength.

EXAMPLE 10

Biotin/Streptavidin Assay

A series of assays were performed to detect streptavidin binding by abiotin receptor layer. A biosensor was first activated with aminochemistry, followed by attachment of a NHS-Biotin linker layer, aspreviously described. Next, 3 μl droplets of streptavidin in PBS wereapplied to the biosensor at various concentrations. The droplets wereallowed to incubate on the biosensor surface for 30 min beforethoroughly washing with PBS rinsing with DI water. The peak resonantwavelength was measured before and after streptavidin binding, and theresonant wavelength shifts are shown in FIG. 38. A linear relationshipbetween peak wavelength and streptavidin concentration was observed, andin this case the lowest streptavidin concentration measured was 0.2μg/ml. This concentration corresponds to a molarity of 3.3 nM.

EXAMPLE 11

Protein-Protein Binding Assay

An assay was performed to demonstrate detection of protein-proteininteractions. As described previously, a biosensor was activated withamino chemistry and an NHS-biotin linker layer. A goat anti-biotinantibody receptor layer was attached to the biotin linker by exposingthe biosensor to a 50 μg/ml concentration solution in PBS for 60 min atroom temperature followed by washing in PBS and rinsing with DI water.In order to prevent interaction of nonspecific proteins with unboundbiotin on the biosensor surface, the biosensor surface was exposed to a1% solution of bovine serum albumin (BSA) in PBS for 30 min. The intentof this step is to “block” unwanted proteins from interacting with thebiosensor. As shown in FIG. 39 a significant amount of BSA isincorporated into the receptor layer, as shown by the increase in peakwavelength that is induced. Following blocking, 3 μl droplets of variousconcentrations of anti-goat IgG were applied to separate locations onthe biosensor surface. The droplets were allowed to incubate for 30 minbefore thorough rinsing with DI water. The biosensor peak resonantwavelength was measured before blocking, after blocking, after receptorlayer binding, and after anti-goat IgG detection for each spot. FIG. 39shows that an anti-goat IgG concentration of 10 μg/ml yields an easilymeasurable wavelength shift.

EXAMPLE 12

Unlabeled ELISA Assay

Another application of a biosensor array platform is its ability toperform Enzyme-Linked Immunosorbent Assays (ELISA) without the need foran enzyme label, and subsequent interaction an enzyme-specific substrateto generate a colored dye. FIG. 40 shows the results of an experimentwhere a biosensor was prepared to detect interferon-γ (IFN-γ) with anIFN-γ antibody receptor molecule. The receptor molecule was covalentlyattached to an NH₂-activated biosensor surface with an SMPT linkermolecule (Pierce Chemical Company, Rockford, Ill.). The peak resonantwavelength shift for application of the NH₂, SMPT, and anti-human IFN-αreceptor molecules were measured for two adjacent locations on thebiosensor surface, as shown in FIG. 40. The two locations were exposedto two different protein solutions in PBS at a concentration of 100μg/ml. The first location was exposed to IFN-γ, which is expected tobind with the receptor molecule, while the second was exposed to neuralgrowth factor (NGF), which is not expected to bind with the receptor.Following a 30 minute incubation the biosensor was measured byilluminating from the bottom, while the top surface remained immersed inliquid. The location exposed to IFN-γ registered a wavelength shift of0.29 nm, while the location exposed to NGF registered a wavelength shiftof only 0.14 nm. Therefore, without the use of any type of enzyme labelor color-generating enzyme reaction, the biosensor was able todiscriminate between solutions containing different types of protein.

EXAMPLE 13

Protease Inhibitor Assay (Caspase-3)

A Caspase-3 protease inhibitor assay was performed to demonstrate thebiosensor's ability to measure the presence and cleavage of smallmolecules in an experimental context that is relevant to pharmaceuticalcompound screening.

Caspases (Cysteine-requiring Aspartate protease) are a family ofproteases that mediate cell death and are important in the process ofapoptosis. Caspase 3, an effector caspase, is the most studied ofmammalian caspases because it can specifically cleave most knowncaspase-related substrates. The caspase 3 assay is based on thehydrolysis of the 4-amino acid peptide substrate NHS-Gly-Asp-Glu-Val-Aspp-nitroanilide (NHS-GDEVD-pNA) by caspase 3, resulting in the release ofthe pNA moiety.

The NHS molecule attached to the N-terminal of the GDEVD provides areactive end group to enable the NHS-GDEVD-pNA complex to be covalentlybound to the biosensor with the pNA portion of the complex oriented awayfrom the surface. Attached in this way, the caspase-3 will have the bestaccess to its substrate cleavage site.

A biosensor was prepared by cleaning in 3:1 H₂SO₄:H₂O₂ solution (roomtemperature, 1 hour), followed by silanation (2% silane in dry acetone,30 sec) and attachment of a poly-phe-lysine (PPL) layer (100 μg/ml PPLin PBS pH 6.0 with 0.5 M NaCl, 10 hours). The NHS-GDEVD-pNA complex wasattached by exposing the biosensor to a 10 mM solution in PBS (pH 8.0,room temperature, 1 hour). A microwell chamber was sealed over thebiosensor surface, and cleavage of pNA was performed by addition of 100μl of caspase-3 in 1× enzyme buffer (100 ng/ml, room temperature, 90minutes). Following exposure to the caspase 3 solution, the biosensorwas washed in PBS. A separate set of experiments using aspectrophotometer were used to confirm the attachment of the complex tothe surface of the biosensor, and functional activity of the caspase-3for removal of the pNA molecule from the surface-bound complex.

The peak resonant frequency of the biosensor was measured beforeattachment of the NHS-GDEVD-pNA complex, after attachment of the complex(MW=860 Da), and after cleavage of the pNA (MW=136) with caspase 3. Asshown in FIG. 41, the attachment of the peptide molecule is clearlymeasurable, as is the subsequent removal of the pNA. The pNA removalsignal of Δλ=0.016 nm is 5.3× higher than the minimum detectable peakwavelength shift of 0.003 nm. The proportion of the added molecularweight and subtracted molecular weight (860 Da/136 Da=6.32) are in closeagreement with the proportion of peak wavelength shift observed for theadded and subtracted material (0.082 nm/0.016 nm=5.14).

The results of this experiment confirm that a biosensor is capable ofmeasuring small peptides (in this case, a 5-mer peptide) without labels,and even detecting the removal of 130 Da portions of a molecule throughthe activity of an enzyme.

EXAMPLE 14

Reaction Kinetics for Protein-Protein Binding Assays

Because a biosensor of the invention can be queried continuously as afunction of time while it is immersed in liquid, a biosensor can beutilized to perform both endpoint-detection experiments and to obtainkinetic information about biochemical reactions. As an example, FIG. 42shows the results of an experiment in which a single biosensor locationis measured continuously through the course of consecutively addingvarious binding partners to the surface. Throughout the experiment, adetection probe illuminated the biosensor through the back of thebiosensor substrate, while biochemistry is performed on the top surfaceof the device. A rubber gasket was sealed around the measured biosensorlocation so that added reagents would be confined, and all measurementswere performed while the top surface of the biosensor was immersed inbuffer solution. After initial cleaning, the biosensor was activatedwith NH₂, and an NHS-Biotin linker molecule. As shown in FIG. 42, goatα-biotin antibodies of several different concentrations (1, 10, 100,1000 μg/ml) were consecutively added to the biosensor and allowed toincubate for 30 minutes while the peak resonant wavelength wasmonitored. Following application of the highest concentration α-BiotinIgG, a second layer of protein was bound to the biosensor surfacethrough the addition of α-goat IgG at several concentrations (0.1, 1,10, and 100 μg/ml). Again, the resonant peak was continuously monitoredas each solution was allowed to incubate on the biosensor for 30minutes. FIG. 42 shows how the resonant peak shifted to greaterwavelength at the end of each incubation period.

FIG. 43 shows the kinetic binding curve for the final resonant peaktransitions from FIG. 42, in which 100 μg/ml of α-goat IgG is added tothe biosensor. The curve displays the type of profile that is typicallyobserved for kinetic binding experiments, in which a rapid increase fromthe base frequency is initially observed, followed by a gradualsaturation of the response. This type of reaction profile was observedfor all the transitions measured in the experiment. FIG. 44 shows thekinetic binding measurement of IgG binding.

The removal of material from the biosensor surface through the activityof an enzyme is also easily observed. When the biosensor from the aboveexperiment (with two protein coatings of goat anti-biotin IgG andanti-goat IgG) is exposed to the protease pepsin at a concentration of 1mg/ml, the enzyme dissociates both IgG molecules, and removes them fromthe biosensor surface. As shown in FIG. 45, the removal of boundmolecules from the surface can be observed as a function of time.

EXAMPLE 15

Proteomics Applications

Biosensors of the invention can be used for proteomics applications. Abiosensor array can be exposed to a test sample that contains a mixtureof binding partners comprising, for example, proteins or a phage displaylibrary, and then the biosensor surface is rinsed to remove all unboundmaterial. The biosensor is optically probed to determine which distinctlocations on the biosensor surface have experienced the greatest degreeof binding, and to provide a quantitative measure of bound material.Next, the biosensor is placed in a “flow cell” that allows a small(e.g., <50 microliters) fixed volume of fluid to make contact to thebiosensor surface. One electrode is activated so as to elute boundmaterial from only a selected biosensor array distinct location. Thebound material becomes diluted within the flow cell liquid. The flowcell liquid is pumped away from the biosensor surface and is storedwithin a microtiter plate or some other container. The flow cell liquidis replaced with fresh solution, and a new biosensor electrode isactivated to elute its bound binding partners. The process is repeateduntil all biosensor distinct locations of interest have been eluted andgathered into separate containers. If the test sample liquid contained amixture of proteins, protein contents within the separate containers canbe analyzed using a technique such as electrospray tandem massspectrometry. If the sample liquid contained a phage display library,the phage clones within the separate containers can be identifiedthrough incubation with a host strain bacteria, concentrationamplification, and analysis of the relevant library DNA sequence.

EXAMPLE 16

Mathematical Resonant Peak Determination

This example discusses some of the findings that have been obtained fromlooking at fitting different types of curves to the observed data.

The first analytic curve examined is a second-order polynomial, given byy=ax ² +bx+cThe least-squares solution to this equation is given by the costfunction

${\phi = {\sum\limits_{i = 1}^{n}\left( {{ax}_{i}^{2} + {bx}_{i} + c - y_{i}} \right)^{2}}},$the minimization of which is imposed by the constraints

$\frac{\partial\phi}{\partial a} = {\frac{\partial\phi}{\partial b} = {\frac{\partial\phi}{\partial c} = 0.}}$Solving these constraints for a, b, and c yields

$\begin{pmatrix}a \\b \\c\end{pmatrix} = {\begin{pmatrix}{\sum x_{i}^{4}} & {\sum x_{i}^{3}} & {\sum x_{i}^{2}} \\{\sum x_{i}^{3}} & {\sum x_{i}^{2}} & {\sum x_{i}} \\{\sum{x2}} & {\sum x_{i}} & n\end{pmatrix}^{- 1} \cdot {\begin{pmatrix}{\sum{x_{i}^{2}y_{i}}} \\{\sum{x_{i}y_{i}}} \\{\sum y_{i}}\end{pmatrix}.}}$The result of one such fit is shown in FIG. 46; the acquired data areshown as dots and the 2^(nd)-order polynomial curve fit is shown as thesolid line.

Empirically, the fitted curve does not appear to have sufficient riseand fall near the peak. An analytic curve that provides bettercharacteristics in this regard is the exponential, such as a Gaussian. Asimple method for performing a Gaussian-like fit is to assume that theform of the curve is given byy=e ^(ax) ² ^(+bx+c),in which case the quadratic equations above can be utilized by formingy′, where y′−ln y. FIG. 46 shows the result of such a fit. The visualappearance of FIG. 46 indicates that the exponential is a better fit,providing a 20% improvement over that of the quadratic fit.

Assuming that the exponential curve is the preferred data fittingmethod, the robustness of the curve fit is examined in two ways: withrespect to shifts in the wavelength and with respect to errors in thesignal amplitude.

To examine the sensitivity of the analytical peak location when thewindow from which the curve fitting is performed is altered to fall 10sampling intervals to the left or to the right of the true maxima. Theresulting shift in mathematically-determined peak location is shown inTable 3. The conclusion to be derived is that the peak location isreasonably robust with respect to the particular window chosen: for ashift of ˜1.5 nm, the corresponding peak location changed by only <0.06nm, or 4 parts in one hundred sensitivity.

To examine the sensitivity of the peak location with respect to noise inthe data, a signal free of noise must be defined, and then incrementalamounts of noise is added to the signal and the impact of this noise onthe peak location is examined. The ideal signal, for purposes of thisexperiment, is the average of 10 resonant spectra acquisitions.

Gaussian noise of varying degrees is superimposed on the ideal signal.For each such manufactured noisy signal, the peak location is estimatedusing the 2^(nd)-order exponential curve fit. This is repeated 25 times,so that the average, maximum, and minimum peak locations are tabulated.This is repeated for a wide range of noise variances—from a variance of0 to a variance of 750. The result is shown in FIG. 47.

TABLE 3 Comparison of peak location as a function of window locationShift Window Peak Location Δ = −10 bins 771.25-782.79 nm 778.8221 nm Δ =0 bins 772.70-784.23 nm 778.8887 nm Δ = +10 bins 774.15-785.65 nm7778.9653 nm

The conclusion of this experiment is that the peak location estimationroutine is extremely robust to noisy signals. The entire range of peaklocations in FIG. 47 is only 1.5 nm, even with as much random noisevariance of 750 superimposed—an amount of noise that is substantiallygreater that what has been observed on the biosensor thus far. Theaverage peak location, despite the level of noise, is within 0.1 nm ofthe ideal location.

Based on these results, a basic algorithm for mathematically determiningthe peak location of a calorimetric resonant biosensor is as follows:

-   -   1. Input data x_(i) and y_(i), i=1, . . . , n    -   2. Find maximum        -   a. Find k such that y_(k)≧y_(i) for all i≠k    -   3. Check that maximum is sufficiently high        -   a. Compute mean y and standard deviation σ of sample        -   b. Continue only if (y_(k)− y)/σ>UserThreshold    -   4. Define curve-fit region of 2w+1 bins (w defined by the user)        -   a. Extract x_(i),k−w≦i≦k+w        -   b. Extract y_(i),k−w≦i≦k+w    -   5. Curve fit        -   a. g_(i)=ln y_(i)        -   b. Perform 2^(nd)-order polynomial fit to obtain g′_(i)            defined on x_(i),k−w≦i≦k+w        -   c. Polynomial fit returns coefficients a,b,c of form            ax²+bx+c        -   d. Exponentiate: y′_(i)=e^(g′i)    -   6. Output        -   a. Peak location p given by x_(p)=−b/2a        -   b. Peak value given by y′_(p)(x_(p))

In summary, a robust peak determination routine has been demonstrated;the statistical results indicate significant insensitivity to the noisein the signal, as well as to the windowing procedure that is used. Theseresults lead to the conclusion that, with reasonable noise statistics,that the peak location can be consistently determined in a majority ofcases to within a fraction of a nm, perhaps as low as 0.1 to 0.05 nm.

EXAMPLE 17

Homogenous Assay Demonstration

An SWS biosensor detects optical density of homogenous fluids that arein contact with its surface, and is able to differentiate fluids withrefractive indices that differ by as little as □n=4×10⁻⁵. Because asolution containing two free non-interacting proteins has a refractiveindex that is different from a solution containing two bound interactingproteins, an SWS biosensor can measure when a protein-proteininteraction has occurred in solution without any kind of particle tag orchemical label.

Three test solutions were prepared for comparison:

-   -   1. Avidin in Phosphate Buffer Solution (PBS), (10 μg/ml)    -   2. Avidin (10 μg/ml)+Bovine Serum Albumin (BSA) (10 μg/ml) in        PBS    -   3. Avidin (10 μg/ml)+Biotinylated BSA (b-BSA) (10 μg/ml) in PBS

A single SWS sensor was used for all measurements to eliminate anypossibility of cross-sensor bias. A 200 μl sample of each test solutionwas applied to the biosensor and allowed to equilibrate for 10 minutesbefore measurement of the SWS biosensor peak resonant wavelength value.Between samples, the biosensor was thoroughly washed with PBS.

The peak resonant wavelength values for the test solutions are plottedin FIG. 51. The avidin solution was taken as the baseline reference forcomparison to the Avidin+BSA and Avidin+b-BSA solutions. Addition of BSAto avidin results in only a small resonant wavelength increase, as thetwo proteins are not expected to interact. However, because biotin andavidin bind strongly (Kd=10⁻¹⁵M), the avidin+b-BSA solution will containlarger bound protein complexes. The peak resonant wavelength value ofthe avidin+b-BSA solution thus provides a large shift compared toavidin+BSA.

The difference in molecular weight between BSA (MW=66 KDa) and b-BSA(MW=68 KDa) is extremely small. Therefore, the differences measuredbetween a solution containing non-interacting proteins (avidin+BSA) andinteracting proteins (avidin+b-BSA) are attributable only to differencesin binding interaction between the two molecules. The bound molecularcomplex results in a solution with a different optical refractive indexthan the solution without bound complex. The optical refractive indexchange is measured by the SWS biosensor.

1. A method of detecting binding of one or more specific bindingsubstances to their respective binding partners comprising: (a)illuminating a biosensor with light and detecting a first peakwavelength value (PWV), wherein the biosensor comprises: (i) atwo-dimensional grating; (ii) a substrate that supports thetwo-dimensional grating; wherein the refractive index of thetwo-dimensional grating is greater than the refractive index of thesubstrate; and (iii) one or more specific binding substances immobilizedon the surface of the two-dimensional grating opposite of the substrate;wherein the one or more specific binding substances do not comprisedetection labels, wherein when the biosensor is illuminated a resonantgrating effect is produced on a reflected radiation spectrum, andwherein the depth and cross-sectional period of the two-dimensionalgrating are less than the wavelength of the resonant grating effect; (b)applying one or more binding partners to the biosensor, wherein thebinding partners do not comprise detection labels; and (c) illuminatingthe biosensor with light and detecting a second PWV; wherein, if the oneor more specific binding substances have bound to their respectivebinding partners, then the second PWV is shifted as compared to thefirst PWV.
 2. The method of claim 1, wherein the one or more specificbinding substances are arranged in an array of distinct locations, andwherein each distinct location of the biosensor is illuminated.
 3. Themethod of claim 1, wherein the two-dimensional grating is comprised of arepeating pattern of shapes selected from the group consisting ofsquares, circles, ellipses, triangles, trapezoids, sinusoidal waves,ovals, rectangles and hexagons.
 4. The method of claim 1, wherein thetwo-dimensional grating is comprised of a material selected from thegroup consisting of zinc sulfide, titanium dioxide, tantalum oxide, andsilicon nitride.
 5. The method of claim 1, wherein the two-dimensionalgrating has a cross-sectional period of about 0.01 microns to about 1micron and a depth of about 0.01 microns to about 1 micron.
 6. Themethod of claim 1, wherein the two-dimensional grating is amineactivated, aldehyde activated, or nickel activated.
 7. The method ofclaim 1, wherein the method further comprises determining the quantityof one or more binding partners bound to the one or more specificbinding substances immobilized on the biosensor by determining thedifference between the first PWV and the second PWV.
 8. The method ofclaim 1, wherein the one or more binding partners comprise one or moretags.
 9. The method of claim 1, wherein the one or more specific bindingsubstances are selected from the group consisting of nucleic acids,polypeptides, antigens, polyclonal antibodies, monoclonal antibodies,single chain antibodies (scFv), F(ab) fragments, F(ab')₂ fragments, Fvfragments, small organic molecules, cells, viruses, bacteria, andbiological sample.
 10. The method of claim 9, wherein the biologicalsample is selected from the group consisting of blood, plasma, serum,gastrointestinal secretions, homogenates of tissues or tumors, synovialfluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinalfluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid,tears, and prostatitc fluid.
 11. The method of claim 1, wherein thebinding partners are selected from the group consisting of nucleicacids, polypeptides, antigens, polyclonal antibodies, monoclonalantibodies, single chain antibodies (scFv), F(ab) fragments, F(ab')₂fragments, Fv fragments, small organic molecules, cells, viruses,bacteria, and biological sample.
 12. The method of claim 11, wherein thebiological sample is selected from the group consisting of blood,plasma, serum, gastrointestinal secretions, homogenates of tissues ortumors, synovial fluid, feces, saliva, sputum, cyst fluid, amnioticfluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen,lymphatic fluid, tears, and prostatitc fluid.
 13. The method of claim 7,wherein the one or more tags are selected from the group consisting ofbiotin, succinimidyl-6-[a-methyl-a-(2-pyridyl-dithio)toluamido]hexanoate (SMPT), dimethylpimelimidate (DMP), and histidine.
 14. Themethod of claim 7, wherein the one or more tags are reacted withstreptavidin, horseradish peroxidase, or streptavidin coatednanoparticles, before step (c).